Insulin-mimetics as therapeutic adjuncts for bone regeneration

ABSTRACT

Methods of promoting bone healing or regeneration by locally administering insulin mimetic agents to patients in need thereof and new uses of insulin-mimetic compounds for accelerating bone-healing processes are disclosed. Bone injury treatment and void filler devices, products and kit suitable for local administration of insulin-mimetic, agents or compositions thereof to patients in need of such treatment are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.14/359.827, filed May 21. 2014, which is the U.S. National Phase ofInternational Application No. PCT/US12/67087, filed Nov. 29, 2012, whichclaims priority under 35 U.S.C. §119(e) to U.S. Provisional PatentApplication Ser. No. 61/564,822, filed on Nov. 29, 2011, and No.61/718,646, filed on Oct. 25, 2012. International Application No.PCT/US12/67087 is a continuation-in-part of International ApplicationNo. PCT/US11/64240, filed on Dec. 9, 2011, which in turn claims priorityunder 35 U.S.C. §119(e) to U.S. Provisional Patent Application Ser. No.61/421,921, filed on Dec. 10, 2010, No. 61/428,342, filed on Dec. 30.2010, and No. 61/454,061, filed on Mar. 18, 2011, all of which arehereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to use of insulin-mimetic agents astherapeutic adjuncts for bone regeneration and methods for bone healingor regeneration in patients by local administration of insulin-mimeticagents..

BACKGROUND OF THE INVENTION

About six million bone fractures, including about 600,000 non-unioncases, occur annually in the United States, :among which approximately10% do not heal. Fracture healing is a complex process that involves thesequential recruitment of cells and the specific temporal expression offactors essential for bone repair. The fracture healing process beginswith the initial formation of a blood clot at the fracture site.Platelets and inflammatory cells within the clot release several factorsthat are important for chemotaxis, proliferation, angiogenesis anddifferentiation of mesenchymal cells into osteoblasts or chondroblasts.

In the orthopedic procedures conducted, about one million performedannually require allograft or autograft. One solution to enhancement ofbone healing is through tissue engineering, in which cells, such asosteoblast, fibroblast, chondroblasts, are treated with bioactivesignaling molecules, e.g., insulin or insulin mimetics, with or withouta carrier such as β-TCP (CaPO₄) and collagen under an appropriateenvironment. Current methods of treatment of bone fractures include (a)electro-stimulation devices (such as PEMF, Exogen) and (b) biologics,such as bone morphogenic proteins (BMPs), e.g., rhBMP-2/ACS (INFUSE®Bone Graft). The latter has been approved by FDA as an autograftreplacement in spine fusion (ALIF) with specific interbody cages (2002),as an adjuvant for repair of tibia fractures with IM nail (2004), andfor craniofacial maxillary surgery (2006), but this method is expensive,costing about $5,000 per application. (Lieberman, J. R., et al., J. BoneJoint Surg. Am., 2002, 84: 1032-1044; Trippel, S. B., et al., J. BoneJoint Surg. Am., 1996, 78: 1272-86.)

The fracture healing process subsequent to the initial hematomaformation can be classified as primary or secondary fracture healing.Primary fracture healing occurs in the presence of rigid internalfixation with little to no interfragmentary strain resulting in directbone formation across the fracture gap. Secondary fracture healingoccurs in response to interfragmentary strain due to an absence offixation or non-rigid fixation resulting in bone formation throughintramembranous and endochondral ossification characterized by responsesfrom the periosteum and external soft tissue.

Intramembranous bone formation originates in the periosteum. Osteoblastslocated within this area produce bone matrix and synthesize growthfactors, which recruit additional cells to the site. Soon after theinitiation of intramembranous ossification, the granulation tissuedirectly adjacent to the fracture site is replaced by cartilage leadingto endochondral hone formation The cartilage temporarily bridging thefracture gap is produced by differentiation of mesenchymal cells intochondrocytes. The cartilaginous callus begins with proliferativechondrocytes and eventually becomes dominated by hypertrophicchondrocytes. Hypertrophic chondrocytes initiate angiogenesis and theresulting vasculature provides a conduit for the recruitment ofosteoblastic progenitors as well as chondroclasts and osteoclasts toresorb the calcified tissue. The osteoblastic progenitors differentiateinto osteoblasts and produce woven hone thereby firming a unitedfracture. The final stages of fracture healing are characterized byremodeling of woven bone to form a structure, which resembles theoriginal tissue and has the mechanical integrity of unfractured bone.

However, the processes of bone metabolism are vastly different from bonerepair. Bone metabolism is the interplay between bone formation and boneresorption. Bone repair, as described previously, is a complex processthat involves the sequential recruitment and the differentiation ofmesenchymal cells towards the appropriate osteoblastic/chondrogeniclineage to repair the fracture/defect site.

Spinal fusion is a common procedure performed for a variety ofconditions including spondylosis, disk disorders, and spinal stenosis.The rates of pseudoarthrosis after single level spinal fusion have beenreported up to 35%. The process of osteogenesis after spinal arthrodesisis similar to that which occurs during fracture healing and heterotopicossification, and agents that increase the rate of fusion have animportant role in decreasing pseudoarthrosis following spinal fusions.To our knowledge, prior to this invention, no in vivo evaluation oftherapy on spinal fusion by local administration of an insulin-mimeticagent, such as a zinc or vanadium compound, has been performed.

There is a clear need to develop new methods for repairing bonefractures by enhancing bone regeneration as well as new methods toenhance spinal fusion.

SUMMARY OF THE INVENTION

The present invention provides a unique strategy for bone regenerationthrough local administration of insulin-mimetic agents, for example, butnot limited to, insulin pathway-stimulating zinc, vanadium, tungsten,molybdenum, niobium, selenium, or manganese compounds.

In one aspect the present invention provides a method of treating a bonecondition in a patient in need of bone regeneration, comprising locallyadministering to the patient a therapeutically effective amount of aninsulin-mimetic agent.

In another aspect the present invention provides use of aninsulin-mimetic compound for manufacture of a medicament foraccelerating bone healing or regeneration in a patient in need thereofcharacterized by local administration of said medicament.

In another aspect the present invention provides a drug delivery deviceor kit, which includes an insulin-mimetic compound and apharmaceutically acceptable carrier, wherein the device or kit isadapted for localized administration of the compound to a patient inneed thereof.

In another inspect the present invention includes localizedadministration of an insulin-mimetic compound or a composition thereofin combination with a second method for promoting bone regeneration,selected from bone autograft methods, bone allograft methods, autologousstein cell treatment methods, methods using autologous growth factorconcentrates, allogeneic stem cell treatment methods, chemicalstimulation methods, electrical stimulation methods, low-intensity pulseultrasound (LIPUS) methods, internal fixation methods, and externalfixation methods.

The present invention also provides a unique strategy to facilitatespinal fusion in spinal fusion procedures.

In one embodiment the present invention provides a bone regenerationmaterial for bone fusion or void filling, comprising an osteoconductivecarrier and an insulin-mimetic agent. In one embodiment, the boneregeneration material contains autograft bone tissue. In anotherembodiment, the bone regeneration material contains allograft bonetissue. In another embodiment, the bone regeneration material containsxenograft bone tissue.

In another aspect the present invention provides a surgical procedurefor stabilizing vertebrae in a spine, including the steps of:

exposing a portion of each of adjacent vertebrae; and

placing supplementary bone tissue material and an insulin-mimetic agentwithin an area between the exposed portions of the adjacent vertebraeand in contact with the exposed portions of both vertebrae;

wherein the insulin-mimetic agent is provided in an amount effective toincrease the rate of fusion of the two vertebrae with the bone tissuematerial.

In one embodiment, the vertebrae are lumbar vertebrae. In anotherembodiment, the vertebrae are cervical vertebrae. In one embodiment, thebone tissue material contains autograft bone tissue. In anotherembodiment, the bone tissue material contains allograft bone tissue. Inone embodiment, the insulin-mimetic agent is mixed with the bone tissuematerial. In a specific embodiment, the bone tissue material isautograft bone tissue and the insulin-mimetic agent is mixed with thebone tissue material after harvesting and before being placed betweenthe exposed potions of the two vertebrae.

In another embodiment, the method further includes the step ofsupporting the two vertebrae with a prosthetic implant configured tostabilize the two vertebrae and promote fusion of the two vertebrae withthe bone tissue material. In one embodiment, the bone tissue contactingsurfaces of the prosthetic implant are coated with the insulin-mimeticagent.

In another aspect, the present invention provides a bone tissue kit forincreasing the rate of fusion of vertebrae in a spinal fusion surgicalprocedure, including the composition containing an insulin-mimetic agentand a pharmaceutically acceptable carrier. In an embodiment the kit alsocontains allograft bone tissue material. In one embodiment theinsulin-mimetic agent and the allograft bone tissue material areprovided in a mixture. In another embodiment, the insulin-mimetic agentand allograft bone tissue material are provided for subsequent mixing.In another aspect the present invention provides a composition forincreasing the rate of spinal fusion in a spinal fusion surgicalprocedure, wherein the composition contains an insulin-mimetic agent anda pharmaceutically acceptable carrier. In one embodiment the compositioncontains allograft bone material

In another aspect, the present invention provides an implantable devicefor enhancing spinal fusion, in which a prosthetic implant is configuredto stabilize and promote the fusion of two adjacent vertebrae, whereinthe bone tissue contacting surfaces of the prosthetic implant are coatedwith a composition comprising an insulin-mimetic agent.

Examples of insulin mimetic agents suitable for the present inventioninclude, but are not limited to, insulin pathway-stimulating zinc,vanadium, tungsten, molybdenum, niobium, selenium, and manganesecompounds.

The present invention thus provides a unique method for promoting bonehealing and enhancing spinal fusion in a patient, preferably mammaliananimal and more preferably a human, either diabetic or non-diabetic.Development of an insulin-mimetic therapy of the present invention wouldobviate the need for developing specialized methods to deliver complexmolecules, such as growth factors like insulin, and thereby reducecosts, eliminate specialized storage, and enhance ease of use. These andother aspects of the present invention will be better appreciated byreference to the following drawings and detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts post-operative X-rays. Representative x-rays takenimmediately post-operative: (A) Einhorn model, (B) model used in thiswork. (Note in (B) the Kirschner wire is going through the trochanter,which helps to stabilize the fracture site and prevent the migration ofthe Kirschner wire.)

FIG. 2 depicts Mechanical Testing Setup: Intact femur before embedded in¾ inch square nut with Field's Metal, where (A) ZINC 10 (3.0 mg/kgZnCl2) and (B) ZINC 8 (1.0 mg/kg ZnCl₂) represent two sets of Zinc,treated femurs harvested 4 weeks post-surgery, showing spiral fractureindicative of healing, compared to (C) ZINC 3 (control) showingnon-spiral fracture indicative of non-union (Left: Intact Femur, Right:Fractured Femur).

FIG. 3 illustrates 4-week radiographs (AP and Medial-Lateral views) ofrepresentative samples of fracture femur bones treated with local ZnCl₂(1.0 and 3.0 mg/Kg) in comparison with saline control.

FIG. 4 illustrates histomorphometry of ZnCl₂ treated fractures incomparison with saline control.

FIG. 5 illustrates 4-week radiographs (AP and Medial-Lateral views) ofrepresentative sample for each group of fractured femur bones treatedwith 1.0 mg/Kg ZnCl₂+CaSO₄ carrier in comparison with CaSO₄ control.

FIG. 6 illustrates comparison of use of ZnCl₂ with the existing therapy(BMP2): (1) a single intramedullary dose (1 mg/kg) of ZnCl₂ with thecalcium sulfate (CaSO₄) vehicle (purple); (2) a single intramedullarydose (3 mg/kg) of ZnCl₂ without a vehicle (green); (3) BMP-2 study useda single percutaneous dose of BMP-2 (80 μg) with buffer vehicle (red);and (4) Exogen study used daily exposure periods of ultrasound treatment(20 mm/day). The average value (duration of 25 days) is shown in blue.

FIG. 7 illustrates 4-week post-fracture radiographs of local manganesechloride (MnCl₂) treatment group vs. saline control.

FIG. 8 illustrates quantification of local VAC levels. Femur bonevanadium concentrations (μg vanadium/gram of bone mass) at one, four,seven, and fourteen days after surgery for fractured and contralateral(intact) femora.

FIG. 9 illustrates histological comparison between VAC and salinecontrol treated rats: Representative sections of saline control, 1.5mg/kg VAC, and 3 mg/kg VAC groups show progression of healing from 10-21days at 1.67× as visualized under stereomicroscope.

FIG. 10 illustrates 4-week radiographs of three representative samplesfor each group of fractured femur hones: (A) saline control, (B) 1.5mg/kg VAC, (C) 3.0 mg/kg VAC.

FIG. 11 illustrates 4-week mechanical testing of treatment with VAC withor without sterilization (normalized to intact femora). The datarepresents average values±standard deviation. * Represent valuesstatistically higher than control, p<0.05 versus saline control.

FIG. 12 illustrates the effect of local vanadium therapy on long-termhealing of femur fractures in normal (non-diabetic) rats, measured byradiographic analysis.

FIG. 13 illustrates comparison of local VAC treatment with current BMP2and Exogen therapies.

FIG. 14 illustrates that the transverse processes of L4-L5 were cleanedof soft tissue, and decorticated with a high-speed burr.

FIG. 15 illustrates that the crushed autograft was then spread over andbetween the transverse processes at the appropriate level (L4-L5). Anequivalent amount of implant or blank was incorporated into theautograft bed.

FIG. 16 illustrates radiographs of the vanadium-treated spines in therat model in comparison with those in the control group.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that insulin-mimeticscan he used to accelerate bone regeneration by stimulating insulinpathway signaling at a fracture site. In particular, the presentinvention is based on the discovery that the biological impact ofinsulin-mimetic agents on bone can be exploited, to play a critical rolein bone healing. Insulin-mimetic agents, such as insulinpathway-stimulating zinc, vanadium, tungsten, molybdemun, niobium,selenium, or manganese compounds, delivered locally with or without acarrier, can improve the torsional strength and bone mineral density ofregenerated bone. Development of a vanadium, zinc, or similar metal salttherapy to accelerate bone regeneration would be beneficialtherapeutically and obviate the need for developing specialized methodsto deliver complex molecules, such as protein growth factors likeinsulin, eliminate specialized storage, enable ease of use, and becost-effective.

The present invention thus uses an insulin-mimetic agent to treatvarious hone conditions, such as bone fractures, and to enhance spinalfusion, for example, in treating spinal arthrodesis. The insulin-mimeticagents suitable for the present invention include, but are not limitedto, insulin pathway-stimulating zinc, vanadium, tungsten, molybdenum,niobium, selenium, or manganese metal or compounds. For example, we usedZnCl₂ alone or as part of a formulation with on orthopedic carrier(CaSO₄, for example) and showed accelerated fracture healing whenapplied directly to the site of fracture post surgery.

Preferably, the patient in need of bone healing is afflicted with a bonecondition selected from bone fracture, bone trauma, arthrodesis,including spinal arthrodesis, extremity arthrodesis and the like, and abone deficit condition associated with post-traumatic bone surgery,post-prosthetic joint surgery, post-plastic bone surgery, post-dentalsurgery, bone chemotherapy treatment, congenital bone defect, posttraumatic bone loss, post surgical bone loss, post infectious bone loss,allograft incorporation or bone radiotherapy treatment.

In another embodiment of this aspect, the bone condition is selectedfrom bone fractures, osseous defects, and delayed unions and non-unions.

Thus, in one aspect, the present invention provides a method ofpromoting bone healing or regeneration in a patient inflicted with abone condition, comprising locally administering to said patient atherapeutically effective amount of an insulin pathway-stimulatinginsulin-mimetic agent.

In one embodiment of this aspect, the insulin-mimetic agent is aninsulin pathway-stimulating zinc, vanadium, tungsten, molybdenum,niobium, selenium, or manganese compound.

In another embodiment of this aspect, the insulin-mimetic agent is azinc, vanadium, or manganese compound.

In another embodiment of this aspect, the insulin-mimetic agent isadministered to the bone injury site.

In another embodiment of this aspect, the method of the presentinvention is used in combination with an allograft method, autograftmethod, xenograft method, alloplastic graft method, or orthopedicbiocomposite method.

In another embodiment of this aspect, the method comprisesco-administering a cytotoxic agent, cytokine or growth inhibitory agentwith said insulin-mimetic agent.

In another embodiment of this aspect, the method is used in conjunctionwith an external bone growth stimulator.

In another embodiment of this aspect, the method comprisesco-administering a bioactive bone agent with the insulin-mimetic agent.

In another embodiment of this aspect, the bioactive bone agent isselected from the group consisting of peptide growth factors,anti-inflammatory factors, pro-inflammatory factors, inhibitors ofapoptosis, MMP inhibitors, and bone catabolic antagonists.

In another embodiment of this aspect, the peptide growth factor isselected from the group consisting of IGF (1,2), PDGF (AA, AB, BB),BMPs, FGF (1-20), TGF-beta (1-3), aFGF, bFGF, EGF, VEGF, parathyroidhormone (PTH), and parathyroid hormone-related protein (PTHrP).

In another embodiment of this aspect, the anti-inflammatory factor isselected from the group consisting of anti-TNFa soluble TNT receptors,IL1ra, soluble IL1 receptors, IL4, IL-10, and IL-13.

In another embodiment of this aspect, the bone catabolic antagonist isselected from the group consisting of bisphosphonates, osteoprotegerin,and statins.

In another embodiment of this aspect, the patient is a mammalian animal.

In another embodiment of this aspect, the patient is a human.

In another embodiment of this aspect, the patient is a non-diabetichuman.

In another aspect, the present invention provides use of aninsulin-mimetic agent for manufacture of a medicament for acceleratingbone healing or regeneration in a patient in need thereof characterizedby local administration of said medicament.

In another aspect, the present invention provides orthopedic and spinalimplants with at least one bone-contacting surface incorporating theinsulin-mimetic compounds and composition of the present invention.Exemplary orthopedic devices include screws, plates, rods, k-wires,pins, hooks, anchors, intramedullary devices, pedicle screws, pediclehooks, spinal fusion cages, spinal fusion plates, prostheses, porousmetal implants such as trabecular metal implants, and the like. Implantssuitable for use with the present invention include metal implantsformed from metals such as titanium, alloys thereof, tantalum, alloysthereof, cobalt chrome alloys, steel alloys, such as stainless steel,and the like. Polymer implants may also be used, including implantsformed from polyglycolic acid (PGA), poly(lactic-co-glycolic acid)(PLGA), polylactic acid (PLA), polycaprolactone (PCL), polyether etherketone (PEEK), polyethylene terephthalate (PET), polypropylene (PP),polycarbonates (PC), poly(ortho esters) (POEs), and the like.

The insulin mimetic may be coated on the bone-contacting surface of theimplant by conventional means. In the alternative the implant may beformulated and fabricated so that the insulin-mimetic is incorporatedinto the bone-contacting surface of the implant. Means by which this canbe accomplished are readily apparent to those of ordinary skill in theart.

In another aspect, the present invention provides a bone injurytreatment kit comprising a therapeutically effective amount of aninsulin-mimetic agent formulated for local administration to a patientinflicted with a bone condition in need of healing or bone regeneration.Such its may also include a device for local administration, such as ahypodermic syringe.

In another aspect, the present invention provides a bone tissuematerial, ceramic bone-grail substitute or mixture thereof forfacilitating bone regeneration or bone fusion. Bone tissue materialsuitable for use in the present invention includes autograft, allograftand xenograft materials.

In one embodiment of this aspect, the bone tissue material contains aninsulin-mimetic agent selected from insulin pathway-stimulating zinc,Vanadium, tungsten, molybdenum, niobium, selenium, and manganesecompounds.

In another embodiment of this aspect, the bone tissue material containsan insulin-mimetic agent selected from vanadium, manganese, and zinccompounds.

In another embodiment of this aspect, the bone tissue material furthercontains a pharmaceutically acceptable carrier.

In another embodiment of this aspect, the pharmaceutically acceptablecarrier is an inorganic salt.

In another embodiment of this aspect, the pharmaceutically acceptablecarrier is an inorganic salt selected from sulfates and phosphates.

In another embodiment of this aspect, the pharmaceutically acceptablecarrier is a calcium salt.

In another aspect, the present invention provides a spinal fusionprocedure utilizing an insulin mimetic agent for enhancing spinalfusion. In one embodiment, a surgical procedure for stabilizingvertebrae in a spine is provided, including the steps of exposing aportion of each of adjacent vertebrae; and placing supplementary bonetissue material, ceramic bone-graft substitute, or mixture thereof, andan insulin-mimetic agent within an area between the exposed portions ofthe adjacent vertebrae and in contact with the exposed portions of bothvertebrae; wherein the insulin-mimetic agent is provided in an amounteffective to increase the rate of fusion of the two vertebrae with thebone tissue material.

In one embodiment of this aspect, the insulin-mimetic agent is a zinc,vanadium, tungsten, molybdenum, niobium, selenium, or manganesecompound.

In another embodiment of this aspect, the insulin-mimetic agent is azinc or vanadium compound.

In another embodiment of this aspect, the insulin-mimetic agent is addedto the supplementary bone tissue material and/or ceramic bone-graftsubstitute to provide a supplementary bone tissue material containingthe insulin-mimetic agent.

In another embodiment of this aspect, the insulin-mimetic agent is addedseparately from the supplementary bone tissue material and/or ceramicbone-graft substitute as a composition further comprising apharmaceutically acceptable carrier.

According to one embodiment, the composition is an insulin-mimeticcalcium sulfate pellet.

In another embodiment of this aspect, the method is in combination withtransplantation of an autograft bone, allograft bone, xenograft bone,ceramic bone-graft substitute, orthopedic biocomposites, and the like.According to one embodiment, an insulin-mimetic agent is admixed withthe autograft, allograft, xenograft ceramic bone-graft substitute,orthopedic biocomposites and the like.

Preferred sites of interest in the patient include sites in need of bonehealing and areas adjacent and/or contiguous to these sites. Optionally,the treatment method of the present invention is combined with at leastone procedure selected from bone autograft, bone allograft methods,methods using autologous growth factor concentrates, autologous stemcell treatment methods, allogeneic stem cell treatment methods, chemicalstimulation methods, electrical stimulation methods, low-intensity pulseultrasound (LIPUS) methods, internal fixation methods, and externalfixation methods, which, in the case of spinal fusion, would stabilizethe fused vertebrae or increase the rate at which the two adjacentvertebrae fuse together.

The insulin-mimetic zinc compounds suitable for the present inventioninclude inorganic zinc compounds, such as mineral acid zinc salts.Examples of inorganic zinc compounds include, but are not limited to,zinc chloride, zinc sulfate, zinc phosphate, zinc, carbonate, and zincnitrate, or combinations thereof.

The insulin-mimetic zinc compounds can also be zinc salts of organicacids. Examples of organic acid zinc salts include, but are not limitedto, zinc acetate, zinc formate, zinc propionate, zinc gluconate,bis(maltolato)zinc, zinc acexamate zinc aspartate,bis(maltolato)zinc(II) [Zu(ma)2], bis(2-hydroxypyridine-N-oxido)zinc(II)[Zn(hpo)2], bis(allixinato)Zn(II) [Zn(alx)2],bis(6-methylpicolinato)Zn(II) [Zn(6mpa)2], bis(aspirinato)zinc(II),bis(pyrrole-2-carboxylato)zinc [Zn(pc)2], bis(alpha-furonic acidato)zinc[Zn(fa)2], bis(thiophene-2-carboxylato)zinc [Zn(tc)2],bis(thiophene-2-acetato)zinc [Zn(ta)2], (N-acetyl-L-cysteinato)Zn(II)[Zn(nac)], zinc(II)/poly(γ-glutamic acid) [Zn(γ-pga)],bis(pyrrolidine-N-dithiocarbamate)zinc(II) [Zn(pdc)₂], zinc(II)L-lactate [Zn(lac)₂], zinc(II) D-(2)-quinic acid [Zn(qui)₂],bis(1,6-dimethyl-3-hydroxy-5-methoxy-2-pentyl-1,4-dihydropyridine-4-thionato)zinc(II)[Zn(tanm)2], β-alanyl-L-histidinato zinc(II) (AHZ), or the like, orcombinations thereof. In another embodiment, the organic acid of zincsalt is a naturally occurring fatty acid.

Suitable organovanadium-based insulin-mimetic agents include, but arenot limited to, vanadyl acetylacetonate (VAC), vanadyl sulfate (VS),vanadyl 3-ethylacetylacetonate (VET), and bis(maltolato)oxovanadium(BMOV), and the like. In a preferred embodiment, the organovanadiumcompound is vanadyl acetylacetonate (VAC). Vanadyl acetylacetonate(VAC), an organic vanadium compound, has demonstrated insulin-mimeticeffects in type 1 and type 2. diabetic animals and human studies andprevented some of the associated complications of diabetes in animalstudies. Additional pharmacological activities of VAC, which have beenstudied, include the inhibition of gluconeogenesis, a decrease inglutamate dehydrogenase activity, and antilipolysis. Use of thesevanadium-based insulin-mimetic agents to accelerate bone healing orregeneration, or as therapeutic adjuncts for cartilage injury and repairbeen disclosed by the present inventors in related U.S. ProvisionalApplication Nos. 61/295,234 and 61/504,777; and PCT Application Nos.PCT/US11/21296 and PCT/US12/45771, which are hereby incorporated byreference in their entirety. Insulin-mimetic vanadium compounds suitablefor use in the present invention include the compounds disclosed in U.S.Pat. Nos, 5,300,496; 5,527,790; 5,688,784; 5,866,563; 5,888,893;6,268,357 and 6,287,586, the disclosures of all of which areincorporated herein by reference.

Suitable tungsten, selenium, molybdenum, niobium, or manganese compoundsas insulin mimetics for bone healing or regeneration are alsoencompassed by the present disclosure, and their forms andadministration modes are within the grasp of an ordinary skill in theart.

Examples of tungsten compounds include, but are not limited to, sodiumtungstate [Na₂WO₄.xH₂O], tungstophosphoric acid [H₃[P(W₃O₁₀)₄].xH₂O],alanine complex of tungstophosphoric acid (WPA-A)[H₃[P(W₃O₁₀)₄][CH₃CH(NH₂)COOH].xH₂O], homo-polyoxotungstates andvanadium polyoxotungstates, tungsten (VI) perooxo complexes (e.g.,(gu)₂[WO₂(O₂)₂] and (gu)[WO(O₂)₂(quin-2-c)], wherein “gu” is guanidiniumand “quin-2-c” is quinoline 2-carboylate), and permetalloxide oftungstate (pW). Molybdenum compounds include, for example,permetalloxide of molybdate.

Niobium compounds include, but are not limited to, Nb(V) peroxocomplexes, e.g., (gu)₃[Nb)(O₂)₄] and (gu)₂[Nb(O₂)₃(quin-2-c), wherein“gu” is guanidinium and “quin-2-c” is quinoline 2-carboylate.

Selenium compounds include, but are not limited to, sodium selenate[Na₂SeO₄.xH2O] and sodium selenite [Na2SeO₃.xH₂O].

Manganese compounds include, but are not limited to,3-O-methyl-D-chiro-inositol+manganese chloride (MnCl₂),D-chiro-inositol+manganese chloride (MnCl₂), manganese sulfate [MnSO4],inositol glycan pseudo-disaccharide Mn(2+) chelate containingD-chiro-inositol 2a (as pinitol) and galactosamine, oral manganese,manganese oxides, e.g., MnO₂, MnOAl₂O₃, and Mn₃O₄.

Other insulin-mimetic metal compounds, in particular, vanadium, zinc,manganese, and tungsten compounds, that may be used for the presentinvention include those disclosed in, for example, Wong. V. V., et al.,Cytotechnology, 2004, 45(3):107-15; and Nomiva, K., et al., J. Inorg.Biochem. 2001, 86(4): 657-667, which are hereby incorporated byreference.

Advantages of small molecules (such as zinc, vanadium, tungsten,molybdenum, niobium, selenium, or manganese)insulin-mimetic agentsinclude, but are not limited to: (a) development of a small moleculeinsulin mimetic can be of great significance to bone fracture patients;(b) insulin composite which requires a carrier may be difficult to meetFDA requirements as a dual agent product; and (c) small molecule insulinmimetics may have longer half life and avoid the storage issues commonlyseen with proteins.

Exemplary healing mechanisms include, but are not limited to: (a)retaining mineralized components in bone, (b) inhibiting release ofmineralized components from bone, (c) stimulating osteoblast activity,(d) reducing osteoclast activity, or (e) stimulating bone remodeling.

The term “therapeutically effective amount,” as used herein, means anamount at which the administration of an agent is physiologicallysignificant. The administration of an agent is physiologicallysignificant if its presence results in a detectable change in the bonehealing process of the patient.

The term “bone injury,” “injured bone,” or the like, as used herein,refers to a bone condition selected from the group consisting of bonefracture, bone trauma, arthrodesis, and a bone deficit conditionassociated with post-traumatic bone surgery, post-prosthetic jointsurgery, post-plastic bone surgery, post-dental surgery, bonechemotherapy treatment, congenital bone loss, post traumatic bone loss,post surgical bone loss, post infectious bone loss, allograftincorporation or bone radiotherapy treatment.

In another embodiment of this aspect, the method is employed in a spinalfusion procedure. Insulin-mimetic compositions of the present inventionare particularly useful adjuncts for spinal fusion procedures. Thecompositions may be used to promote vertebral fusion and spinalstablization and also to improve function of spinal stabilizationdevices.

According to one embodiment, an interbody device, which is a prostheticimplant configured to stabilize two adjacent vertebrae and promotefusion of the two vertebrae, is provided, wherein the bone tissuecontacting surfaces of the prosthetic implant are the device coated witha composition comprising an insulin-mimetic agent. The device may alsobe configured to supply autograft bone, allograft bone, xenograft bone,ceramic bone-graft substitutes, orthopedic biocomposites, or the like,to the exposed surfaces of the two adjacent vertebrae, which bone orhone-graft substitute may or may not be admixed with an insulin-mimeticagent.

In another aspect, the present invention provides a bone tissue kit forfacilitating fusion of vertebrae in a spinal fusion surgical procedure,including a composition containing an insulin-mimetic agent and apharmaceutically acceptable carrier. In an embodiment the kit alsocontains allograft bone tissue material, xenograft bone tissue material,and/or ceramic bone-graft substitute. In one embodiment theinsulin-mimetic agent and the allograft bone tissue material, xenograftbone tissue material, and/or ceramic bone-graft substitute are providedin a mixture. In another embodiment, the insulin-mimetic agent andallograft bone tissue material, xenograft bone tissue material, orceramic bone-graft substitute are provided for subsequent mixing.

In one embodiment of this aspect, the insulin-mimetic agent is selectedfrom insulin pathway-stimulating zinc, vanadium, tungsten, molybdenum,niobium, selenium, and manganese compounds, and combinations thereof.The insulin-mimetic agent can be in any form known in the art that issuitable for use in spinal fusion procedures.

In another aspect, the present invention provides a compositioncomprising an insulin-mimetic, agent for enhancing spinal fusion in aspinal fusion surgical procedure, wherein the composition contains aninsulin-mimetic agent and a pharmaceutically acceptable carrier. In oneembodiment, the composition contains allograft bone material and/orceramic bone-graft substitute.

In one embodiment of this aspect, the insulin-mimetic agent is selectedfrom insulin pathway-stimulating zinc, vanadium, tungsten, molybdenum,niobium, selenium, and manganese compounds, and combinations thereof.

In one embodiment of this aspect, the implantable device is combinedwith autograft, allograft, or synthetic bone void fillers (e.g. ceramic)in order to enhance posterior or posterolateral fusion of the cervical,thoracic or lumbar spine. This involves decortication of the native hostbone of the lamina or lateral masses (posterior fusion) or the side ofthe facet joints and transverse processes (posterolateral fusion). Thebone grafting mixture (including the insulin-mimetic compound) are thenpacked over these prepared areas to induce segmental fusion.

In another embodiment of this aspect, the implantable device is combinedwith autograft, allograft, or synthetic (ceramic) bone void filler inthe central chamber of an interbody device to enhance fusion between thevertebral bodies of the anterior column of the spine (anterior interbodyspinal fusion). This is performed after anterior discectomies anddecompressions as well as after anterior corpectomies when the vertebralbody is removed for purposes of decompression or to address trauma,tumor or infection involving the vertebral body.

In another embodiment of this aspect, an insulin-mimetic agent is usedas a surface modification to an interbody device (cage) inserted betweenthe vertebral bodies of the anterior column of the spine to effect ananterior interbody spinal fusion. Such cages are used to reconstruct theanterior column of the spine after discectomy or corpectomy (see above).The areas requiring surface modification would be the surfaces that willbe in apposition to the corresponding vertebral endplates of thesegments cephalad (above) and caudal (below).

In another embodiment of this aspect, an insulin-mimetic agent is usedas a surface modification to spinal fixation devices such as pediclescrews, inserted by either open or percutaneous posterior approach. Suchscrews are placed by drilling a pilot hole that extends down through thepedicle and into the vertebral body in a posterior-to-anteriordirection. The screws in each vertebral body are then connected to eachother by rods to stabilize the spanned motion segments.

In another embodiment of this aspect, an insulin-mimetic agent is usedas a surface modification to spinal fixation devices such as anteriorvertebral body screws used in conjunction with plates, inserted by openor minimally invasive anterior or anterolateral approaches. Suchanterior vertebral body screws are typically placed in ananterior-to-posterior direction in the cervical and lower lumbar spine.In the upper lumbar and thoracic spine they are often placed into thevertebral body from an anterolateral starting point.

In any of the embodiments of this aspect, the insulin-mimetic agent isselected from zinc, vanadium, tungsten, molybdenum, niobium, selenium,or manganese compounds, and combinations thereof, preferably a vanadium,manganese, or zinc compound, for example, VAC, manganese chloride, orzinc chloride.

Examples of diseases or conditions that make a patient in need of spinalfusion include, but are not limited to, arthrodesis, degenerative discdisease, spinal disc herniation, discogenic pain, spinal tumor,vertebral fracture, scoliosis, kyphosis (i.e., Scheuermann's disease),spondylolisthesis, spondylosis, Posterior Rami Syndrome, otherdegenerative spinal conditions, and any other conditions that causeinstability of the spine.

It will be appreciated that actual preferred amounts of a pharmaceuticalcomposition used in a given therapy will vary depending upon theparticular form being utilized, the particular compositions formulatedthe mode of application, and the particular site of administration, andother such factors that are recognized by those skilled in the artincluding the attendant physician or veterinarian. Optimaladministration rates for as given protocol of administration can bereadily determined by those skilled in the art using conventional dosagedetermination tests.

Dosages of an insulin-mimetic suitable for the present invention mayvary depending on the particular use envisioned. The determination ofthe appropriate dosage or route of administration is well within theskill of an ordinary physician. The dosage regimen for theinsulin-mimetic agents of the present invention will vary depending uponknown factors, such as the pharmacodynamic characteristics of theparticular agent, and its mode and route of administration; the species,age, sex, health, medical condition, and weight of the recipient; thenature and extent of the symptoms; etc. For exmple, the local dosage ofa particular insulin-mimetic agent, such as a zinc, vanadium, ormanganese compound, may depend more on the bone condition than on theweight of a patient. A dosage of local administration may significantlydiffer from a dosage of systemic administration, and a dosage ofadministration in a solution form may differ from a dosage when it isadministered through the surface coating on an implantable device.Without being bound by any particular theory, the dosage of aninsulin-mimetic agent according to the present invention should be at alevel so that the insulin pathway in a patient is stimulated in order toaccelerate the bone healing or regeneration process.

By way of general guidance, the dosage of each active ingredient, whenused for the indicated effects, will range between about 0.001 to about200 mg/Kg based on a patient's weight, preferably between about 0.01 toabout 100 mg/Kg, and most preferably between about 0.1 to about 50mg/Kg. The doses can be repeated whenever needed, or considered to bebeneficial to the bone healing and regeneration processes as determinedby a physician, for example, once daily, once weekly, once every otherweek, once monthly, or any other time period that may provide mostbenefits to a particular patient.

The route of administration of “local zinc” via “insulin mimeticdelivery system” is in accordance with known methods, e.g. viaimmediate-release, controlled-release, sustained-release, andextended-release means. Preferred modes of administration for theinsulin-mimetic delivery system include injection directly intoafflicted bone or a fusion site and areas adjacent and/or contiguous tothese sites, or surgical implantation of insulin-mimetic agent(s)directly into the fusion sites and area adjacent and/or contiguous tothese sites. This type of system will allow temporal control of releaseas well as location of release as stated above.

The formulations used herein may also contain more than one activecompound as necessary for the particular indication being treated,preferably those with complement-ary activities that do not adverselyaffect each other. Alternatively, or in addition, the formulation maycomprise a cytotoxic agent, cytokine or growth inhibitory agent. Suchmolecules are present in combinations and amounts that are effective forthe intended purpose.

Vanadium, which exists in +4 (vanadyl) and +5 (vanadate) compounds inthe biological body, have demonstrated poor absorption rates within thegastrointestinal (GI) tract and GI side-effects, such as diarrhea andvomiting. As a result, additional organic vanadium compounds, i.e.,vanadyl 3-ethylacetylacetonate (VET), bis(maltolato)oxo-vanadium (BMOV),and VAC, have been synthesized in order to improve absorption andsafety. VAC with an organic ligand has been proven to be more effectivein its anti-diabetic function compared with other vanadium compounds,including BMOV, VS, and VET.

Therapeutic formulations of vanadium compounds in the vanadium deliverysystems employable in the methods of the present invention are preparedfor storage by mixing the vanadium compound having the desired degree ofpurity with optional pharmaceutically acceptable carriers, excipients,or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol,A. Ed. (1980)). Such therapeutic formulations can be in the form oflyophilized formulations or aqueous solutions. Acceptable biocompatiblecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and may include buffers, forexample, phosphate, citrate, and other organic acids; antioxidantsincluding ascorbic acid and methionine; preservatives (e.g.octadecyldimethylbenzyl ammonium chloride; hexa-methonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens, for example, methyl or propyl paraben;catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); lowmolecular weight (less than about 10 residues) polypeptides; proteins,for example, serum albumin, gelatin, or immunoglobulins, hydrophilicpolymers, for example, polyvinylpyrrolidone; amino acids, for example,glycine, glutamine asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, dextrins, or hyaluronan; chelating agents, forexample, EDTA, sugars, for example, sucrose, mannitol, trehalose orsorbitol; salt-forming counter-ions, for example, sodium; metalcomplexes (e.g. Zn-protein complexes); and/or non-ionic surfactants, forexample, TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

In order for the formulations to be used for in vivo administration,they must be sterile. The formulation may be readily rendered sterile byfiltration through sterile filtration membranes, prior to or followinglyophilization and reconstitution. The therapeutic formulations hereinpreferably are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

The vanadium may also be entrapped in microcapsules prepared, forexample by coacervation techniques or by interfacial polymerization, forexample, hydroxy-methyl-cellulose or gelatin-microcapsules andpoly-(methylmethacrylate) microcapsules, respectively. Such preparationscan be administered in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th Edition (or newer), Osol A.Ed. (1980).

Optionally, the organovanadium agent in the vanadium delivery systemsincludes a porous calcium phosphate, non-porous calcium phosphate,hydroxy-apatite, tricalcium phosphate, tetracalcium phosphate, calciumsulfate, calcium minerals obtained from natural bone, inorganic bone,organic bone, or a combination thereof.

Where sustained-release or extended-release administration of vanadiumin the vanadium delivery systems is desired, microencapsulation iscontemplated. Microencapsulation of recombinant proteins for sustainedrelease has been successfully performed with human growth hormone (rhGH,interferon-α, -β, -γ (rhIFN-α,-β,-γ), interleukin-2, and MN rgp120.Johnson et al., Nat. Med. 2: 795-799 (1996): Yasuda, Biomed. Ther. 27:1221-1223 (1993): Hora et al., Bio/Technology 8: 755-758 (1990);Cleland, “Design and Production of Single Immunization Vaccines UsingPolylactide Polyglycolide Microsphere Systems” in Vaccine Design: TheSubunit and Adjuvant Approach, Powell and Newman, eds., (Plenum Press:New York, 1995), pp. 439-462: WO 97/03692, WO 9640072, WO 96/07399 andU.S. Pat. No. 5,654,010.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing thevanadium in the vanadium delivery systems, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include one or more polyanhydrides (e.g.,U.S. Pat. Nos. 4,891,225; 4,767,628), polyesters, for example,polyglycolides, polylactides and polylactide-co-glycolides (e.g., U.S.Pat. No. 3,773,919; U.S. Pat. No. 4,767,628; U.S. Pat. No. 4,530,840;Kulkarni et al., Arch. Surg. 93: 839 (1966)), polyamino acids, forexample, polylysine, polymers and copolymers of polyethylene oxide,polyethylene oxide acrylates, polyacrylates, ethylene-vinyl acetates,polyamides, polyurethanes, polyorthoesters, polyacetylnitriles,polyphosphazenes, and polyester hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinyl-alcohol)), cellulose,acyl substituted cellulose acetates, non-degradable polyurethanes,polystyrenes, polyvinyl chloride, polyvinyl fluoride,poly(vinylimidazole), chlorosulphonated polyolefins, polyethylene oxide,copolymers of L-glutamic acid and .gamma.-ethyl-L-glutamate,non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolicacid copolymers, for example, the LUPRON DEPOT™ (injectable microspherescomposed of lactic acid-glycolic acid copolymer and leuprolide acetate),and poly-D-(−)-3-hydroxybutyric acid. While polymers such asethylene-vinyl acetate and lactic acid-glycolic acid enable release forover 100 days, certain hydrogels release proteins for shorter timeperiods. Additional non-biodegradable polymers which may be employed arepolyethylene, polyvinyl pyrrolidone, ethylene vinylacetate, polyethyleneglycol, cellulose acetate but rate and cellulose acetate propionate.

Alternatively, sustained-release formulations may be composed ofdegradable biological materials, for example, collagen and derivativesthereof, bioerodible fatty acids (e.g., palimitic acid, steric acid,oleic acid, and the like). Biodegradable polymers are attractive drugformulations because of their biocompatibility, high responsibility forspecific degradation, and ease of incorporating the active drug into thebiological matrix. For example, hyaluronic acid (HA) may be crosslinkedand used as a swellable polymeric delivery vehicle for biologicalmaterials. U.S. Pat. No. 4,957,744: Valle et al., Polym. Mater. Sci.Eng., 62: 731-735 (1991). HA polymer grafted with polyethylene glycolhas also been prepared as an improved delivery matrix which reduced bothundesired drug leakage and the denaturing associated with long termstorage at physiological conditions. Kazuteru, M., J. Controlled Release59:77-86 (1999). Additional biodegradable polymers which may be used arepoly(caprolactone), polyanhydrides, polyamino acids, polyorthoesters,polycyanoacrylates, poly(phosphazines), poly(phosphodiesters),poly-esteramides, polydioxanones, polyacetals, polyketals,polycarbonates, polyortho-carbonates degradable and nontoxicpolyurethanes, polyhydroxylbutyrates, polyhydroxy-valerates,polyalkylene oxalates, polyalkylene succinates, poly(malic acid),chitin, and chitosan.

Alternatively, biodegradable hydrogels may be used as controlled-releasematerials for the vanadium compounds in the vanadium delivery systems.Through the appropriate choice of macromers, membranes can be producedwith a range of permeability, pore sizes and degradation rates suitablefor different types of vanadium compounds in the vanadium deliverysystems.

Alternatively sustained-release delivery systems for vanadium in thevanadium delivery systems can be composed of dispersions. Dispersionsmay further be classified as either suspensions or emulsions. In thecontext of delivery vehicles for a vanadium compound, suspensions are amixture of very small solid particles which are dispersed (more or lessuniformly) in a liquid medium. The solid particles of a suspension canrange in size from a few nanometers to hundreds of microns, and includemicrospheres, microcapsules and nanospheres. Emulsions, on the otherhand, are a mixture of two or more immiscible liquids held in suspensionby small quantities of emulsifiers. Emulsifiers form an interfacial filmbetween the immiscible liquids and are also blown as surfactants ordetergents. Emulsion formulations can be both oil in water (o/w) whereinwater is in a continuous phase while the oil or fat is dispersed, aswell as water in oil (w/o), wherein the oil is in a continuous phasewhile the water is dispersed. One example of a suitablesustained-release formulation is disclosed in WO 97/25563. Additionally,emulsions for use with a vanadium compound in the present inventioninclude multiple emulsions, microemulsions, microdroplets and liposomes.Micro-droplets are unilamellar phospholipid vesicles that consist of aspherical lipid layer with an oil phase inside. E.g., U.S. Pat. No.4,622,219 and U.S. Pat. No. 4,725,442. Liposomes are phospholipidvesicles prepared by mixing water-insoluble polar lipids with an aqueoussolution.

Alternatively, the sustained-release formulations of vanadium in thevanadium delivery systems may be developed using poly-lactic-coglycolicacid (PLGA), a polymer exhibiting a strong degree of biocompatibilityand a wide range of biodegradable properties. The degradation productsof PLGA, lactic and glycolic acids, are cleared quickly from the humanbody. Moreover, the degradability of this polymer can be adjusted frommonths to years depending on its molecular weight and composition. Forfurther information see Lewis, “Controlled Release of Bioactive Agentsfrom Lactide/Glycolide polymer,” in Biogradable Polymers as DrugDelivery Systems M. Chasin and R. Langeer, editors (Marcel Dekker: NewYork, 1990), pp. 1-41.

As an illustrated example, an insulin-mimetic may be continuouslyadministered locally to a site via a delivery pump. In one embodiment,the pump is worn externally (in a pocket or on the belt) and attached tothe body with a long, thin, and flexible plastic tubing that has aneedle or soft cannula (thin plastic tube), and the cannula or needle isinserted and then left in place beneath the skin. The needle or cannulaand tubing can be changed, for example, every 48 to 72 hours. The pumpwould store the insulin-mimetic in a cartridge and release it based onthe optimal delivery rate. Optionally, the pump is programmed to give asmall dose of a drug continuously through the day and night, which incertain circumstances may be preferred.

Various applications of the present invention are listed in Table 1.

TABLE 1 Applications of local administration of insulin-mimetics.Application Method Examples Fracture Healing Local delivery Bonefractures treated by to closed or closed reduction or open fracturessurgical reduction Fracture Non-unions Local delivery to Treatment offractures that non-healing have experienced delayed fractures or failedhealing Arthrodesis/fusion Injection to Treatment of spine fusion (e.g.,spine, fusion increase of joints such as foot osteogenesis and ankles,wrist) in certain joints Allograft incorporation Injection withinTreatment of intercalary and around allograft defect after traumaticused to fill defect injuries, tumor resection, failed arthroplasty, etc.

The compounds and compositions of the present invention are effectiveinsulin mimetics without the issues associated with biologics such asinsulin. They have various notable advantages over biologics, forexample, their high tolerance to manufacturing process and conditions(e.g., elevated temperatures). In the case of ZnCl₂ it is a known,highly stable compound commonly used in medical products, has a longshelf life, and has no storage and contamination/sterilization relatedissues.

The compounds and compositions of the present invention are alsoversatile—they can be used directly or as part of a formulation with acarrier applied to the site of fracture to accelerate fracture healing.No special techniques need to be developed in order to use theinventions described. For example, zinc compounds can be applieddirectly to the fracture site as part of the surgery or intramedullary.

Similarly, vanadium-based insulin mimetics can accelerate fracturehealing process (fracture healing resolved in 4-5 weeks), reduced timeto recovery in both normal and diabetic patients), resolve non-healingfractures (10% of annual total), resolve diabetic (compromised hostmodel) fractures, in addition to a wide array of applications in severalsectors of orthopedic devices. In the case of the vanadium surfacemodification approach, vanadium can be used to modify existing implants(plates, nails, screws, k-wires, etc.) to potentiate osseous healing.

Similar to the zinc compounds, vanadium compounds are also effectiveinsulin mimetics without the issues associated with biologics such asinsulin. They have the following advantages over biologics, for example,the ability to tolerate manufacturing process (for example, elevatedtemperatures), the high stability and long shelf life, and no storageand contamination/sterilization issues. Moreover, the disclosed vanadiumcompounds are versatile—they can be used directly or as part of aformulation with a carrier applied to the site of fracture to acceleratefracture healing. Surfaces of materials commonly used in orthopedicimplants can be modified with vanadium and such modified materials werealso shown to be effective in accelerating fracture healing. No specialtechniques need to be developed in order to use the inventionsdescribed. In the case of vanadium compounds, the material can beapplied directly to the fracture site as part of the surgery orpercutaneously injected. In the case of the surface modified implants,standard surgical techniques associated with the implants can be used.In the present studies disclosed, the quality of the bone formation wascharacterized using X-ray, micro-CT scans as well as measuringmechanical parameters such as torque, rigidity, shear modulus and shearstress, and in all cases, the quality of the healed bone was compared tothat of normal bone in the same animal.

When an implantable device coated by a composite surface coatingcomprising an insulin-mimetic compound of the present invention is used,the coating can be formed by any methods known in the relevant art, forexample, without limitation, those disclosed in Petrova. R. andSuwattananont, N., J. Electr. Mat., 34(5):8 (2005)). For example,suitable methods include chemical vapor deposition (CVD), physical vapordeposition (PVD), thermochemical treatment, oxidation, and plasmaspraying (Fischer, R. C., Met. Progr. (1986); Habig, K. H., Tribol.Int., 22:65 (1989)). A suitable coating of the present invention mayalso comprise combinations of multiple, preferably two or three, layersobtained by forming first boron diffusion coating followed by CVD (Z.Zakhariev, Z., et al. Surf Coating Technol., 31:265 (1987)).Thermochemical treatment techniques have been well investigated and usedwidely in the industry. This is a method by which nonmetals or metalsare penetrated by thermodiffusion followed by chemical reaction into thesurface. By thermochemical treatment, the surface layer changes itscomposition, structure, and properties.

Other suitable coating techniques may include, but are not limited to,carburizing, nitriding, carbonitriding, chromizing, and aluminizing.Among these coating techniques, boronizing, being a thermochemicalprocess, is used to produce hard and wear-resistant surfaces. As aperson of ordinary skill in the art would understand, different coatingtechniques may be used to make the vanadium-based coatings and coateddevices of the present invention in order to have desired propertiessuitable for specific purposes.

The present invention also finds wide application in veterinarymedicines to treat a variety of factures or enhance spinal fusion in amammalian animal, including but not limited to, horses, dogs, cats, orany other domestic or wild mammalian animals. A particular usefulapplication may be found, for example, in treating an injured racehorse.

The following non-limiting examples illustrate certain aspects of theinvention.

EXAMPLES Example 1 Use of Zinc Compounds to Accelerate Bone FractureHealing Materials and Methods The BB Wistar Rat Model Animal Source andOrigin

Diabetic Resistance (DR) BB Wistar rats used in the study were obtainedfrom a breeding colony at UMDNJ-New Jersey Medical School (NJMS). Therats were housed under controlled environmental conditions and fed adlibitum. All research protocols were approved by the InstitutionalAnimal Care and Use Committee at University of Medicine and Dentistry ofNew Jersey-New Jersey Medical School.

Diabetic Resistant BB Wistar Rats

A total of 24 DR BB Wistar rats were utilized in the study. Due tounstable fixation during mechanical testing, three samples were removed.Another sample was removed due to complications associated with apost-operative infection. The remaining 17 animals were used formechanical testing and were distributed between the control saline(n=6), 0.1 mg/kg zinc chloride (n=2), 1.0 mg/kg zinc chloride (n=3), 3.0mg/kg zinc chloride (n=3), 6.0 mg/kg zinc chloride (n=4) and 10.0 mg/kgzinc chloride (n=3) groups.

Closed Femoral Fracture Model

Surgery was performed in DR animals between ages 93 and 99 days using aclosed mid-diaphyseal fracture model, on the right femur as describedpreviously.

General anesthesia was administrated by intraperitoneal (IP) injectionof ketamine (60 mg/kg) and xylazine (8 mg/kg). The right leg of each ratwas shaved and the incision site was cleansed with Betadine and 70%alcohol. An approximately 1 cm medial, parapatellar skin incision wasmade over the patella. The patella was dislocated laterally and theinterchondylar notch of the distal femur was exposed. An entry hole wasmade with an 18 gauge needle and the femur was reamed with the 18 gaugeneedle. A Kirschner wire (316LVM stainless steel, 0.04 inch diameter.Small Parts, Inc., Miami Lakes, Fl.) was inserted the length of themedullary canal, and drilled through the trochanter of the femur. Thekirschner wire was cut flush with the femoral condyles. Afterirrigation, the wound was closed with 4-0 vicryl resorbable suture. Aclosed midshaft fracture was then created unilaterally with the use of athree-point bending fracture machine. X-rays were taken to determinewhether the fracture was of acceptable configuration. An appropriatefracture is an approximately mid-diaphyseal, low energy, transversefracture (FIG. 1). The rats were allowed to ambulate freely immediatelypost-fracture. This closed fracture model is commonly used to evaluatethe efficacy of osseous wound healing devices and drugs.

Local Zinc Delivery

Zinc Chloride [(ZnCl₂) Sigma Aldrich, St. Louis, Mo.] mixed with abuffer was injected into the intramedullary canal prior to fracture. Thebuffer consisted of sodium acetate, sodium chloride methylhydroxybenzoate, and zinc chloride. Doses of 1.0 mg/kg and 3.0 mg/kgzinc chloride were tested and administered at a volume of 0.1 mL.

Mechanical Testing

Fractured and contralateral femora were resected at three and four weekspost-fracture. Femora were cleaned of soft tissue and the intramedullaryrod was removed. Samples were wrapped in saline (0.9% NaCl) soaked gauzeand stored at −20° C. Prior to testing, all femora were removed from thefreezer and allowed to thaw to room temperature for three to four hours.The proximal and distal ends of the fractured and contralateral femorawere embedded in ¾ inch square nuts with Field's Metal, leaving anapproximate gauge length of 18 mm (FIG. 2). After measuring callus,gauge length and femur dimensions, torsional testing was conducted usinga servohydraulics machine (MTS Systems Corp., Eden Prairie, Minn.) witha 20 Nmm reaction torque cell (Interface, Scottsdale, Ariz.) and testedto failure at a rate of 2.0 deg/sec. The maximum torque to failure andangle to failure were determined from the force to angular displacementdata.

Maximum torque to failure, maximum torsional rigidity, shear modulus,and maximum shear stress were calculated through standard equations(Ekeland, A. et al., Acta Orthop. Sand., 1981, 52(6):605-13: Engesaeter,L. B. et al., Acta Orthop. Scand., 1978, 49(6):512-8). Maximum torque tofailure and maximum torsional rigidity are considered extrinsicproperties while shear modulus and maximum shear stress are consideredintrinsic properties. Maximum torque to failure was defined as the pointwhere an increase in angular displacement failed to produce any furtherincrease in torque. Maximum torsional rigidity is a function of themaximum torque to failure, gauge length (distance of the exposed femurbetween the embedded proximal and distal end) and angular displacement.Maximum shear stress is a function of the maximum torque to failure,maximum radius within the mid-diaphyseal region and the polar moment ofinertia. The polar moment of inertia was calculated by modeling thefemur as a hollow ellipse. Engesaeter et al. (1978) demonstrated thatthe calculated polar moment of inertia using the hollow ellipse modeldiffered from the measured polar moment of inertia by only two percent(Engesaeter, L. B., et al., Acta Orthop, Scand., 1978, 49(6):512-8).

In order to compare the biomechanical parameters between differenttreatment groups, the data was normalized by dividing each fracturedfemur value by its corresponding intact, contralateral femur value (FIG.2). Normalization was used to minimize biological variability due todifferences in age and weight among rats.

In addition to the biomechanical parameters determined through torsionaltesting, the mode of failure can also provide substantial information.The mode of torsional failure as determined by gross inspection providedan indication as to the extent of healing. A spiral failure in themid-diaphyseal region indicated a complete union while a transversefailure through the fracture site indicated a nonunion. A combinationspiral/transverse failure indicated a partial union (FIG. 2).

Data and Statistical Analysis

Analysis of variance (ANOVA) was performed followed by Holm-Sidakpost-hoc tests to determine differences between the treated ZnCl₂ groupswith a group size larger than two. A Student's t-test was performed toidentify differences between the two treated groups in the ZnCl₂ study(SigmaStat 3.0, SPSS Inc., Chicago, Ill.). A P value less than 0.05 wasconsidered statistically significant.

General Description of Animal Surgery

A closed mid-diaphyseal fracture surgery was performed on the rightfemur of each rat as described previously. (Beam. H. A., et al., J.Orthop. Res. 2002, 20(6):1210-1216, Gandhi, A., et at. Bone 2006,38(4)540-546.) General anesthesia was administered by intraperitonealinjection of ketamine (60 mg/kg) and xylazine (8 mg/kg). A closed,midshaft fracture was then created using a three-point bending fractureinstrument (BBC Specialty Automotive, Linden N.J.) and confirmed withX-rays immediately post-fracture.

Preparation of ZnCl₂ Solution

Zinc chloride (ZnCl₂), Sigma Aldrich, St. Louis, Mo., mixed with sterilewater at various doses with or without a calcium sulfate carrier, wereinjected into the intramedullary canal prior to fracture. Doses of ZnCl₂were not based on each animal's body weight, but on a lowertheoretically tolerable dose for a 290-gram BB Wistar rat, which wouldnot elicit heavy metal poisoning or behavioral changes. This weight isover 50 grams lower than the average weight of non-diabetic BB Wistarrats at an age of approximately 90 days (the age of investigation inthis study). A 0.1 ml volume of the ZnCl₂ solution was administeredlocally via a single injection into the marrow space for each doseexamined.

Preparation of ZnCl₂/CaSO₄ Formulation

To prepare the ZnCl₂/CaSO₄ mixture, CaSO₄ (2 g) were placed in glassvials. The vials were placed in an autoclave and sterilized at for twohours in a dry cycle. CaSO₄ powder (0.8 g) was mixed with 400 μl ofsaline or 400 μl of ZnCl₂ solution (1.0 mg/kg) for one minute at roomtemperature. The mixture was packed into the barrel of a 1 cc sterilesyringe and pushed down into the open orifice of the syringe barrel byinsertion of the syringe plunger. After attaching an 18-gauge sterileneedle to the syringe barrel, 0.1 ml volume of the mixture was directlyinjected into the rat femoral canal (non-diabetic BB Wistar rat) priorto Kirschner wire insertion and fracture.

Microradiographic Evaluation

Serial microradiographs were obtained from all animals every two weeksafter surgery. Under the same anesthesia as described above, the ratswere positioned prone and lateral and anteroposterior (AP) radiographsof their femurs were obtained. Radiographs were taken using a PackardFaxitron (MX 20-Radiographic Inspection System) and Kodak MinR-2000mammography film. Exposures were for 30 seconds at 55 kVp. Magnifiedradiographs were obtained of resected femurs. Qualitative analysis wasperformed on all radiographic sample at four weeks post-fracture. Twoindependent observers individually scored radiographs based on bridgingof the lateral and AP femoral orientations. Treatment group averageswere computed to estimate healing at 4 weeks post-fracture. The analysiswas conducted in a blinded fashion using a validated, five-pointradiographic scoring system, 0=no evident bony bridging, 1=bony bridgingof one cortex, 2=bony bridging of two cortices, 3=bony bridging of threecortices, and 4=bony bridging of all four cortices. (See Bergenstock, M.W., et al., J. Orthop. Trauma 2005, 19(10):717-723.)

Torsional Mechanical Testing

Torsional testing was conducted at four weeks using a servohydraulicsmachine (MTS Sys. Corp., Eden Prairie, Minn.) with a 20 Nm reactiontorque cell (Interface, Scottsdale, Ariz.). Femurs were tested tofailure at a rate of 2.0 deg/sec at four and six week time points. Thepeak torque, torsional rigidity, effective bulk modulus, and theeffective maximum shear stress (σ) were determined with standardequations that model each femur as a hollow ellipse. (Ekeland A., etal., Acta Orthop. Scand. 1981, 52(6):605-613 ; Engesaeter, L. B., al.,Acta Orthop. Scand. 1978, 49(512-518). In order to compare thebiomechanical parameters between different groups, the data wasnormalized by dividing each fractured femur value by its correspondingintact, contralateral femur value. Torsional mechanical testing islimited by differences in gauge length during bone potting in Field'smetal. Placement and dimension of fracture gap can contribute tostandard deviations. Finally, this test is limited because it relies ona mathematical model that assumes the femur is a hollow ellipse, asopposed to the natural architecture of femoral bone. (Levenston, M. E.et al., J. Bone Miner. Res. 1994, 9(9):1459-1465.)

Early-Stage Healing Analysis by Histomorphometry

The fractured femora were resected at seven days post-fracture,decalcified, dehydrated, embedded in paraffin, and sectioned usingstandard histological techniques. Sections were stained with Masson'sTrichrome (Accustain™ Trichrome Staining kit, Sigma Diagnostics, St.Louis, Mo.) for histological observation using an Olympus BH2-RFCAmicroscope (Olympus Optical Co., Ltd., Shinjuku-ku, Tokyo, Japan).Digital images were collected using a Nikon DXM1200F digital camera(Nikon, Tokyo, Japan). Cartilage, new bone, and total callus area weremeasured from the digital images using Image-Pro Plus software (version5, Media Cybernetics, Inc., Silver Spring, Md.). Total cartilage and newhone area were normalized to total callus area and expressed as thepercent area. Two independent reviewers were used to minimizeinconsistencies.

Late-Stage Healing Analysis by Histomorphometry

To examine the effects of VAC at later stages of fracture healing,femora were resected from animals in the groups described above at day21, embedded and sectioned using standard histological techniques. Thisincludes dehydration, soaking in Xylenes, and finally pre-embedding in alayer of Polymethylmethacrylate (PMMA). After embedding in pure PMMA andallowed to solidify in a hot water bath, slides were sectioned from thePMMA blocks, polished, and stained with a combination of Stevenel's blueand Van Gieson picro-fuchsin (SVG). Histological images of fracturecalluses were obtained using an Olympus SZX12 upright microscope(Olympus Optical Co, LTD, Japan) connected via a CCD camera (Optronics,Goleta, Calif.) to a personal computer and analyzed with the Bioquantsoftware package (Biometrics, Inc, Nashville, Tenn.). Parameters thatwere compared include a) callus area, b) percent calcified tissue area,and c) percent cartilage area. Limitations of this procedure includeproduction of slides with high thicknesses, due to the difficultiesassociated with sectioning PMMA. This limits the number of possiblesections that may be cut for staining in addition to analysis ofcellular morphology, due to overlapping layers of cells.

General Health of Animals

The age of the BB Wistar rats at the time of fracture surgery variedbetween 75 and 137 days. However, animals amongst treatment groups wereage and sex matched for each experiment. The percent weight changefollowing surgery to the day of sacrifice was similar amongst treatmentgroups.

RESULTS General Health

In this experiment, the rats were 93-117 days old at time of fracture.No significant difference in percent weight gain was found betweentreatment groups from time of fracture until euthanization (Table 2).Blood glucose levels were higher in the zinc chloride treated rats, butthe blood glucose values were within the normal range for all treatmentgroups (Table 2).

TABLE 2 General health of non-DM BB Wistar rats: local zinc (ZnCl₂)delivery without a carrier (Mechanical Testing) Blood Glucose (mg/dl) 12Hours Post-Surgery % Weight gain Saline Control (n = 6) 81.7 ± 4.3 ^(a)3.5 ± 2.3 0.1 mg/kg ZnCl₂ 87.0 ± 7.1 ^(a) 15.3 ± 11.5 (n = 2) 1.0 mg/kgZnCl₂ 99.3 ± 3.1 ^(b) 11.0 ± 9.4  (n = 3) 3.0 mg/kg ZnCl₂ 105.0 ± 4.4^(b)   6.9 ± 11.7 (n = 3) 6.0 mg/kg ZnCl₂ 88.0 ± 4.3 ^(a) 4.6 ± 2.3 (n =4) 10.0 mg/kg ZnCl₂ 87.7 ± 8.5 ^(a) 4.2 ± 2.0 (n = 3) The datarepresents average values ± standard deviation ^(a) represents valuessignificantly less than the 3.0 mg/kg ZnCl₂ group; p < 0.05 ^(b)represents values significantly less than the saline group; p < 0.05

Microradiographic Evaluation

At four weeks post-fracture, femurs from rats treated with ZnCl₂ hadsignificantly higher radiograph scores than control femurs (Table 3).

Mechanical Testing Results Local ZnCl₂ (No Carrier)

The effect of local zinc therapy on healing of femur fractures wasmeasured by torsional mechanical testing. At four weeks post-fracture,rats treated with local ZnCl₂ displayed improved mechanical propertiesof the fractured femora compared to the untreated group. Radiographstaken at 4 weeks post-fracture support this finding (FIG. 3). Table 3represents the radiograph scoring values at two different dosages.

TABLE 3 Radiographic scoring evaluation 4 Weeks Post-Fracture (# ofcortices bridged) Saline Control (n = 6) 1.2 ± 0.75 (n = 6) 1.0 mg/kgZnCl₂ (n = 3) 3.0 ± 0.6* (n = 3) 3.0 mg/kg ZnCl₂ (n = 3) 3.3 ± 0.6* (n =3) The data represents average values ± standard deviation *Representvalues statistically higher than control, p < 0.05

Table 4 summarizes the results of the mechanical testing of the bone forfractured bone, following four weeks of healing. The effective shearstress was 1.6× and 2.2× higher at four weeks post-fracture for thehealing femurs from the ZnCl— treated animals, at dosages of 1.0 mg/kgand 3.0 mg/kg respectively. When normalized to their intact,contralateral femurs, the percent maximum torque to failure, percenttorsional rigidity, and percent effective shear modulus, of thefractured femora were 2.0×, 3.8×, and 8.0× higher, respectively, at thedosage of 3 mg/kg ZnCl₂ compared to the control group (p<0.05).

The effect of local zinc therapy on healing of femur fractures in normal(non-diabetic) rats was measured by torsional mechanical testing. At 4weeks post-fracture, fractured femurs from the rats treated with zincchloride had greater mechanical properties than the fractured femursfrom the control group. For the 10 mg/kg ZnCl₂ group, the maximumtorsional rigidity was significantly greater than the untreated group(Table 4). When the mechanical parameters of the fractured femora werenormalized to the intact, contralateral femora, percent maximum torqueto failure (saline group vs. 3 mg/kg ZnCl₂ group p<0.05), torsionalrigidity (saline group vs. 3 mg/kg ZnCl₂ group p<0.05), and shearmodulus (Saline group vs. 3 mg/kg ZnCL₂ group p<0.05, Saline group vs.10 mg/kg ZnCL₂ group p<0.05) were significantly greater in the localzinc treated groups when compared to the saline group (Table 4).

Healing was assessed by radiographic examination and quantified bymechanical testing. Local ZnCl₂ treatment improved radiographicappearance and significantly increased the mechanical strength offractured femurs. At four weeks post-fracture, the average percentmaximum torque to failure of the fractured femora for 3.0 mg/kg ZnCl₂was significantly (2.04 times) greater (82.0% of contralateral vs.27.0%), compared to the untreated saline group. Percent maximumtorsional rigidity values for 3.0 mg/kg ZnCl₂ was significantly (3.85times) greater (97.0% of contralateral vs. 20.0%), compared to theuntreated saline group. Percent shear modulus values for both low (3.0mg/kg ZnCl₂) and high (10.0 mg/kg ZnCl₂) doses were significantlygreater, with high dose 8.8 times greater (36.0% of contralateral vs.4.0%), and low dose 9.0 times greater (39.0% of contralateral vs. 4.0%)compared to the untreated saline group. The data indicate that localZnCl treatment enhanced bone regeneration during fracture healing andindicates that zinc and potentially similar metals can be used astherapeutically as osteogenic drugs.

TABLE 4 Four weeks post-fracture mechanical testing with local zinc(ZnCl₂) Fractured Femur Values Maximum Effective Torque to MaximumTorsional Effective Shear Stress Shear Failure (Nmm) Rigidity (Nmm²/rad)Modulus (MPa) (MPa) Saline Control 161 ± 48 9.9 × 10³ ± 4.7 × 10³ 2.6 ×10² ± 1.1 × 10² 17 ± 4  (n = 6) 0.1 mg/kg ZnCl₂ 252 ± 13 2.1 × 10⁴ ± 4.2× 10³ 1.7 × 10³ ± 3.3 × 10² 61 ± 14  (n = 2) 1.0 mg/kg ZnCl₂ 281 ± 862.2 × 10⁴ ± 2.7 × 10³ 9.7 × 10² ± 3.6 × 10² 44 ± 15  (n = 3) 3.0 mg/kgZnCl₂ 369 ± 74 3.1 × 10⁴ ± 1.1 × 10⁴ 1.3 × 10³ ± 6.4 × 10² 55 ± 21* (n =3) 6.0 mg/kg ZnCl₂  276 ± 190 2.9 × 10⁴ ± 1.6 × 10⁴ 1.1 × 10³ ± 7.5 ×10² 32 ± 25* (n = 4) 10.0 mg/kg ZnCl₂ 254 ± 36 3.6 × 10⁴ ± 2.5 × 10⁴ 3.0 × 10³ ± 1.9 × 10³* 62 ± 30  (n = 3) Fractured Femur ValuesNormalized to the Contralateral (Intact) Femur Percent Percent MaximumPercent Maximum Percent Effective Effective Torque to Failure TorsionalRigidity Shear Modulus Shear Stress Saline Control 27 ± 18 20 ± 10 4 ± 210 ± 5  (n = 6) 0.1 mg/kg ZnCl₂ 57 ± 12 87 ± 14 34 ± 4  33 ± 14 (n = 2)1.0 mg/kg ZnCl₂ 65 ± 29 55 ± 14 32 ± 15 18 ± 8  (n = 3) 3.0 mg/kg ZnCl₂ 82 ± 25*  97 ± 55*  36 ± 10* 27 ± 17 (n = 3) 6.0 mg/kg ZnCl₂ 38 ± 20 62± 35 18 ± 12 15 ± 10 (n = 4) 10.0 mg/kg ZnCl₂ 41 ± 8  73 ± 44  39 ± 23*27 ± 11 (n = 3) The data represents average values ± standard deviation*Represents values statistically higher than saline control, p < 0.05versus saline control. One way ANOVA between 6 groups (all pairwise)with a Holm-Sidak post-hoc analysis

Histomorphometry of Zinc Chloride Treated Fractures

The results of histomorphometry of zinc chloride treated fractures after7, 10, and 21 days are listed in Table 5 and illustrated in FIG. 4.

TABLE 5 Histomorphometry of zinc chloride-treated fractures % Bone %Cartilage 7 Day Saline Control (n = 5)  8.08 ± 2.45 3.00 ± 1.7 3.0 mg/kg(n = 7)  18.92 ± 5.97 *  4.64 ± 3.41 10 Day Saline Control (n = 5) 17.90± 5.20 16.3 ± 2.8 3.0 mg/kg (n = 7) 21.31 ± 5.40 12.79 ± 3.02 21 DaySaline Control (n = 6) 25.00 ± 6.10  6.1 ± 3.2 3.0 mg/kg (n = 7) 24.47 ±3.53 11.57 ± 5.53Local ZnCl₂/CaSo₄ Formulations

We repeated the above experiment with formulations of ZnCl₂/CaSO₄applied to the fracture site. Radiographs taken at four weekspost-fracture support this finding (FIG. 5) shows significant boneformation.

TABLE 6 Four weeks post-fracture mechanical testing with formulation ofzinc chloride (ZnCl₂) with CaSO₄ carrier applied to the fracture site.Fractured Femur Values Maximum Torque Maximum Torsional Effective ShearEffective Shear to Failure (Nmm) Rigidity (Nmm2/rad) Modulus (MPa)Stress (MPa) Saline Control (n = 6) 161 ± 48  9.9 × 10³ ± 4.7 × 10³ 2.6× 10² ± 1.1 × 10² 17 ± 4  CaSO₄ Control (n = 7) 251 ± 78  2.1 × 10⁴ ±1.3 × 10⁴ 6.0 × 10² ± 3.7 × 10² 26 ± 10 0.5 mg/kg ZnCl2 + CaSO₄ (n = 4)337 ± 175 3.0 × 10⁴ ± 7.9 × 10³ 1.1 × 10² ± 9.4 × 10² 36 ± 22 1.0 mg/kgZnCl2 + CaSO₄ (n = 7)  396 ± 112*  3.9 × 10⁴ ± 1.4 × 10⁴*^(,#)  1.3 ×10³ ± 7.1 × 10²*  46 ± 16* 3.0 mg/kg ZnCl2 + CaSO₄ (n = 5) 262 ± 126 2.1× 10⁴ ± 7.8 × 10³ 7.0 × 10² ± 3.1 × 10² 33 ± 19 Fractured Femur CaluesNormalized to the Contralateral (Intact) Femur Percent Maximum Percentmazimum Percent Effective Percent Effective Torque to Failure TorsionalRigidity Shear Modulus Shear Stress Saline Control (n = 6) 27 ± 18 20 ±10 4 ± 2 10 ± 5  CaSO₄ Control (n = 7) 48 ± 21 55 ± 35 11 ± 7  16 ± 7 0.5 mg/kg ZnCl2 + CaSO₄ (n = 4) 56 ± 31 63 ± 20 17 ± 19 19 ± 12 1.0mg/kg ZnCl2 + CaSO₄ (n = 7)  75 ± 18*  79 ± 32* 18 ± 10 27 ± 8* 3.0mg/kg ZnCl2 + CaSO₄ (n = 5) 45 ± 22 52 ± 22 14 ± 8  20 ± 14 The datarepresents average values ± standard deviation *Represents valuesstatistically higher than saline control, p < 0.05 versus salinecontrol. ^(#)Represents values statistically higher than CaSO4 control,p < 0.05 versus CaSO4 control. One-way ANOVA between 5 groups withHolm-Sidak post-hoc analysis

Table 6 summarizes the results of the mechanical testing of the bone forfractured bone, following four weeks of healing using the formulation.The effective shear stress was 2.7× and 1.7× higher at four weekspost-fracture for the healing femurs from the ZnCl₂/CaSO₄ treatedanimals, at dosages of 1.0 mg/kg compared to saline and CaSO₄ control,respectively. When normalized to their intact, contralateral femurs, thepercent maximum torque to failure, percent torsional rigidity, andpercent effective shear modulus, of the fractured femora were 2.8×,4.0×, and 4.5× higher, respectively, at the dosage of 1 mg/kg ZnCl₂CaSO₄ compared to the saline control group (p<0.05).

Comparison of Use of ZnCl₂ with Existing Therapy (BMP2)

As an insulin-mimetic adjunct, zinc compounds can be used to acceleratebone regeneration by stimulating insulin signaling at the fracture site.ZnCl₂ treatment applied directly to the fracture site significantlyincreased the mechanical parameters of the bone in treated animals afterfour weeks, compared to controls. It accelerated fracture-healingprocess (fracture healing resolved in four to five weeks, instead ofaverage eight to ten weeks in standard rat femur fracture model).

Other healing adjuncts currently approved for FDA use in the UnitedStates include Bone Morphogenic Proteins (BMP's) and Exogen/PulsedElectromagnetic Fields (PEMF). However, BMPs may be associated withshortcomings such as causing ectopic bone growth and having high costper application; and Exogen/PEMF therapy has shown only limited provenusefulness in fracture healing and needs for patient compliance fordaily use.

The chart in FIG. 6 compares the use of ZnCl₂ (alone or in combinationwith CaSO₄) with the currently approved products (BMP-2 and Exogen) forfracture healing. Each of these studies examined the effectiveness of atherapeutic adjunct on femur fracture healing by measuring the maximumtorque to failure at the four week time point.

Specifically the following were compared to their repective untreatedcontrol group: (1) a single intramedullary dose (1 mg/kg) of ZnCl2 withthe calcium sulfate (CaSO4) vehicle (purple); (2) a singleintramedullary dose (3 mg/kg) of ZnCl2 without a vehicle (green); (3)BMP-2 study used a single percutaneous dose of BMP-2 (80 mg) with buffervehicle (red) (see Einhorn, T. A., et al., J. Bone Joint Surg. Am. 2003,85-A(8):1425-1435); and (4) Exogen study used daily exposure periods ofultrasound treatment (20 mm/day). The average value (duration of 25days) is shown in blue (see Azuma, Y., et al., J. Bone Miner. Res. 2001,16(4):671-680.

As graphically shown, use of single application of insulin-mimetic likezinc chloride results in significantly increased improvement of torqueto failure and other mechanical properties of the fracture callus,compared to the existing gold standard of LIPUS and BMP2, usingtorsional mechanical testing of rat femur fracture model of Bonnarrensand Einhorn.

In summary, we have found that acute, local ZnCl₂ treatment (eitheralone or as a formulation with a carrier), administered immediatelyprior to an induced fracture, promoted healing in non-diabetic rats. Atthe four week time point, mechanical parameters of the healed bone weresubstantially higher than that of the control group. This is consistentwith our earlier findings of insulin's ability to promote bone growthwhen applied to the fracture site. This is also consistent with ourfinding that insulin mimetic compounds such as vanadyl acetylacetonate(VAC) accelerate fracture healing much like insulin. Though also aninsulin mimetic, unlike VAC. ZnCl₂ is a compound commonly used in manycommercial medical products and hence potential regulatory barriers areminimal. This suggests that insulin mimetics applied locally to thefracture may be used therapeutically as a fracture-healing adjunct, andlocal ZnCl₂ treatment is a cost-effective fracture-healing adjunct andhas potential for other possible orthopedic applications.

The above preliminary data indicate that local treatment with aninsulin-mimetic such as zinc is an effective method to enhance boneregeneration. Mechanical parameters and radiography revealed that bonebridged at four weeks after fracture in the zinc-treated rats ascompared to saline treated controls. Spiral fractures that occurredduring mechanical testing support the radiographic observations andsuggest that local ZnCl₂ application at the dosages tested mayaccelerate fracture healing, compared to untreated controls. These datasupport additional testing of ZnC₂ as a therapeutic agent to accelerateor enhance bone regeneration.

Example 2 Use of Manganese Compounds for Fracture Healing Material andMethods Rat Model

The animal model used for this study is the Diabetes Resistant (DR) BBWistar Rat. It will be obtained from a breeding colony at UMDNJ-NewJersey Medical School (NJMS) which is maintained under controlledenvironmental conditions and fed ad libitum.

The BB Wistar colony was established from diabetic-prone BB Wistar ratsoriginally obtained from BioBreeding (Toronto, Canada). Similar to humantype I diabetes, spontaneously diabetic BB Wistar rats display markedhyperglycemia, glycosuria and weight loss within a day of onset,associated with decreased plasma insulin after undergoing selective andcomplete destruction of pancreatic β-cells. If left untreated, diabeticBB Wistar rats would become ketoacidic within several days, resulting indeath. Genetic analysis of the BB-Wistar rat shows the development ofdiabetes is strongly related to the presence of the iddm4 diabetogenicsuceptibility locus on chromosome 4 as well as at least four other locirelated to further susceptibility and the development of lymphopenia(Martin, A. M., et al., Diabetes 1999, 48(11):2138-41).

The DR-BB Wistar rat colony was also originally purchased fromBioBreeding and has been established as an effective control group forstudies involving the diabetic BB Wistar rat. Under controlledenvironmental conditions, DR-BB Wistar rats would never developspontaneous type I diabetes, are non-lymphopenic, and areimmunocompetent. It has since been used in our lab as a model of a“normal” rat model. The choice was made to utilize the DR-BB Wistar rat,rather than purchase commercially available rats for our studies,because of the ability to expand the colony by breeding at any time asnecessary for different protocols, as well our familiarity with the ratover years of its utilization in similar protocols. The consistent useof the BB Wistar and the DR-BB Wistar rat models allow for an increasein reliability when comparing data between our various protocols.

General Health of Animals

The age of the BB Wistar rats at the time of fracture surgery variedbetween 95 and 137 days. However, animals amongst treatment groups wereage and sex matched for each experiment. The percent weight changefollowing surgery to the day of sacrifice was similar amongst treatmentgroups.

Surgical Technique

Surgery will be performed to produce a closed mid-diaphyseal fracturemodel in the right femur. General anesthesia will be administered priorto surgery by intraperitoneal (IP) injection of ketamine (60 mg/kg) andxylazine (8 mg/kg). The right leg of each rat is shaved and the incisionsite is prepared with Betadine and 70% alcohol. A one centimeter medial,parapatellar skin incision is made, followed by a smaller longitudinalincision through the quadriceps muscle, just proximal to the quadricepstendon. The patella is dislocated laterally and the intercondylar notchof the distal femur is exposed. An entry hole is made with an 18-guageneedle and the femoral intramedullary canal is subsequently reamed. Forexperimental groups, 0.1 mL of MnCl2 solution(of different dosage) isinjected into the medullary canal of the femur. For control groups, 0.1mL of saline is injected. A Kirschner wire (316LVM stainless steel, 0.04inch diameter, Small Parts, Inc., Miami Lakes, Fla.) is inserted intothe intramedullary canal. The Kirschner wire is cut flush with thefemoral condyles. After irrigation, the wound is closed with 4-0 vicrylresorbable sutures. A closed midshaft fracture is then createdunilaterally with the use of a three-point bending fracture machine.X-rays are taken to determine whether the fracture is of acceptableconfiguration. Only transverse, mid-diaphyseal fractures are accepted.The rats are allowed to ambulate freely immediately post-fracture.

Post Surgery Procedures

X-rays are taken at two-week intervals to the day of euthanasia. Aftereuthanasia x-rays are taken as well. To take x-rays, animals will begiven a half dose of anesthesia. All groups will be monitored closelyfor four days after surgery for infection, and the ability to ambulatefreely.

Torsional Mechanical Testing

Torsional testing was conducted at 4 weeks post-fracture, using aservohydraulics machine (MTS Sys. Corp., Eden Prairie, Minn.) with a 20Nm reaction torque cell (Interface, Scottsdale, Ariz.), Femurs weretested to failure at a rate of 2.0 deg/sec at four weeks post-fracture.The peak torque, torsional rigidity, effective bulk modulus, and theeffective maximum shear stress (σ) were determined with standardequations that model each femur as a hollow ellipse (Ekeland. A. et al.,Acta Orthop, Scand. 1981, 52(6):605-613; Engesaeter, L. B. et al., ActaOrthop. Scand 1978, 49(6):512-518). In order to compare thebiomechanical parameters between different groups, the data wasnormalized by dividing each fractured femur value by its correspondingintact, contralateral femur value. Torsional mechanical testing islimited by differences in gauge length during bone potting in Field'smetal. Placement and dimension of fracture gap can contribute tostandard deviations. Finally, this test is limited because it relies ona mathematical model that assumes the femur is a hollow ellipse, asopposed to the natural architecture of femoral bone (Levenston, M. E.,et al., J. Bone Miner. Res. 1994, 9(9):1459-1465).

Early-Stage Healing Analysis by Histomorphometry

The fractured femora were resected at seven and ten clays post-fracture,decalcified, dehydrated, embedded in paraffin, and sectioned usingstandard histological techniques. Sections were stained with Masson'sTrichrome (Accustain™ Trichrome Staining kit, Sigma Diagnostics, St.Louis, Mo.) for histological observation using an Olympus BH2-RFCAmicroscope (Olympus Optical Co., Ltd., Shinjuku-ku, Tokyo, Japan).Digital images were collected using a Nikon DXM1200F digital camera(Nikon, Tokyo, Japan). Cartilage, new bone, and total callus area weremeasured from the digital images using Image-Pro Plus software (version5, Media Cybernetics, Inc., Silver Spring, Md.). Total cartilage and newbone area were normalized to total callus area and expressed as thepercent area. Two independent reviewers were used to minimizeinconsistencies.

Data and Statistical Analysis

Analysis of variance (ANOVA) was performed followed by Holm-Sidakpost-hoc tests to determine differences between the treated MnCl₂ groupswith a group size larger than two. A Student's t-test was performed toidentify differences between the two treated groups in the MnCl₂ study(SigmaStat 3.0, SPSS Inc., Chicago, Ill.). A p value less than 0.05 wasconsidered statistically significant,

Results Mechanical Testing Local MnCl₂ No Carrier

The effect of local MnCl2 therapy on healing of femur fractures wasmeasured by torsional mechanical testing. At four weeks post-fracture,rats treated with MnCl₂ displayed improved mechanical properties of thefractured femora compared to the saline control group. The maximumtorque to failure was significantly increased compared to the salinecontrol group (p<0.05: 0.125 mg/kg MnCl₂, p<0.05: 0.25 mg/kg MnCl₂,p<0.05: 0.3 mg/kg MnCl₂) (Table 7). When the mechanical parameters ofthe fractured femora were normalized to the intact, contralateralfemora, percent torsional rigidity was significantly greater in thelocal MnCl₂ treated groups when compared to the saline control group(p<0.05: 0.125 mg/kg MnCl₂, p<0.05: 0.25 mg/kg MnCl₂)) (Table 7).

TABLE 7 Four weeks post-fracture mechanical testing with local manganesechloride (MnCl₂) Fractured Femur Values Maximum Maximum Effective Torqueto Torsional Rigidity Effective Shear Shear Stress Failure (Nmm)(Nmm²/rad) Modulus (MPa) (MPa) Saline Control 161 ± 48 9.9 × 10³ ± 4.7 ×10³ 2.6 × 10² ± 1.1 × 10² 17 ± 4 (n = 6) 0.083 mg/kg 272 ± 39 2.6 × 10⁴± 1.2 × 10⁴ 8.7 × 10² ± 4.9 × 10² 30 ± 8 MnCl₂ (n = 5) 0.125 mg/kg  351± 59* 4.2 × 10⁴ ± 1.1 × 10⁴ 6.4 × 10² ± 8.8 × 10¹ 21 ± 6 MnCl₂, (n = 4)0.25 mg/kg  344 ± 84* 3.4 × 10⁴ ± 1.6 × 10⁴ 8.1 × 10² ± 5.0 × 10²  32 ±11 MnCl₂ (n = 4) 0.30 mg/kg,   323 ± 135* 3.0 × 10⁴ ± 2.6 × 10⁴ 7.6 ×10² ± 9.2 × 10²  27 ± 23 MnCl₂ (n = 6) 0.50 mg/kg 230 ± 83 2.9 × 10⁴ ±1.2 × 10⁴ 6.2 × 10² ± 3.5 × 10² 19 ± 9 MnCl₂ (n = 6) Fractured FemurValues Normalized to the Contralateral (Intact) Femur Percent PercentPercent Maximum maximum Percent Effective Effective Torque to FailureTorsional Rigidity Shear Modulus Shear Stress Saline Control  27 ± 18 20± 10 4 ± 2 10 ± 5 (n = 6) 0.083 mg/kg 42 ± 5 56 ± 30 8 ± 7  8 ± 4 MnCl₂(n = 5) 0.125 mg/kg 54 ± 5 103 ± 40* 16 ± 11 14 ± 5 MnCl₂ (n = 4) 0.25mg/kg  55 ± 19  80 ± 34* 14 ± 9  16 ± 6 MnCl₂ (n = 4) 0.30 mg/kg  50 ±22 50 ± 37 10 ± 12  16 ± 12 MnCl₂ (n = 6) 0.50 mg/kg  38 ± 15 61 ± 16 17± 13 14 ± 7 MnCl₂ (n = 6) The data represents average values ± standarddeviation *Represents values statistically higher than saline control, p< 0.05 versus saline control.

Radiographic Analysis

Radiographs taken at four weeks post-fracture support these mechanicaltesting results (FIG. 7). At four weeks, the fractures treated with 0.25mg/kg dosage of MnCl₂ displayed increased mineralized tissue than salinecontrols. analysis of radiographs showed the MnCl₂ group demonstratedunion at the subperiosteal bony area and at the callus, whereas salinecontrol radiographs had no evidence of union.

Histomorphometric Analysis

In animals treated with MnCl2, histomorphometric analysis revealed astatistically lower (p<0.05) percent cartilage in 0.3 mg/kg Mn Cl2treated femora, compared to controls at seven days (Table 8). At tendays, percent mineralized tissue in 0.3 mg/kg Mn Cl2treated femora weresignificantly increased (p<0.05: 0.3 mg/kg Mn Cl2) compared to salinecontrols (Table 8).

TABLE 8 Histology: comparison of manganese chloride with saline control7 days post fracture 10 days post fracture Group % cartilage % new bone% cartilage % new bone Saline 6.116 ± 2.51 15.668 ± 2.93 9.542 ± 1.0214.011 ± 1.29   0.3  2.859 ± 1.09 ^(#) 15.604 ± 2.39 11.051 ± 3.05 18.866 ± 2.28 * mg/kg * Represents values statistically higher thansaline control, p < 0.001 ^(#) Represents values statistically lowerthan saline control, p < 0.05

Example 3 Use of Vanadium Compounds for Bone Fracture Healing MethodGeneral Description of Animal Surgery

A closed mid-diaphyseal fracture surgery was performed on the rightfemur of each rat as described previously. General anesthesia wasadministered by intraperitoneal injection of ketamine (60 mg/kg) andxylazine (8 mg/kg). A closed, midshaft fracture was then created using athree-point bending fracture instrument (BBC Specialty Automotive,Linden N.J.) and confirmed with X-rays immediately post-fracture.

Preparation of VAC Solution

Vanadyl acetylacetonate (VAC), Sigma Aldrich, St. Louis, Mo., mixed withsterile water at various doses with or without a calcium sulfatecarrier, were injected into the intramedullary canal prior to fracture.VAC was chosen over alternative organo-vanadium compounds such as BMOVand VS, due to its' observed superior potency at stimulating proteinkinase B (PKB). Glycogen synthase kinase 3 beta (GSK-3β), and proteintyrosine phosphorylation (PTP). Additionally, Mehdi et al. noted morepotent Insulin receptor beta subunit (IRβ), and Insulin receptorsubstrate 1 (IRS-1) tyrosine phosphorylation, for VAC, compared to BMOVand VS. Doses of VAC were not based on each animal's body weight, but ona lower theoretically tolerable dose for a 290 gram BB Wistar rat, whichwould not elicit heavy metal poisoning or behavioral changes. Thisweight is over 50 grams lower than the average weight of non-diabetic BBWistar rats at an age of approximately 90 days (the age of investigationin this study). The daily subcutaneous dose injected by Zhang et al. (3mg VAC/kg body weight) was multiplied by this average weight of 0.29 kg.A 0.1 mL volume of the VAC solution was administered locally via asingle injection into the marrow space for each dose examined. Thisreduced the absolute concentration of VAC administered in the high doseto the same concentration as Zhang et al., while the 1.5 mg/kg dose was50% of the dose administered by Zhang. Later the 0.5 mg/kg dose (33.3%of low dose) and 0.25 mg/kg dose (16.6% of low dose) were evaluated todetermine the optimal dose of VAC, and examine the range ofeffectiveness of VAC.

Preparation of VAC/CaSO₄ Formulation

To prepare the CaSO₄-VAC mixture, two grams of CaSO₄ were placed inglass vials. The vials were placed in an autoclave and sterilized at fortwo hours in a dry cycle. CaSO₄ powder (0.8 grams) was mixed with 400 μlof saline or 400 μl of VAC solution (0.25 mg/kg and 1.5 mg/kg) for oneminute at room temperature. The mixture was packed into the barrel of a1 cc sterile syringe and pushed down into the open orifice of thesyringe barrel by insertion of the syringe plunger. After attaching an18-gauge sterile needle to the syringe barrel, 0.1 ml volume of themixture was directly injected into the rat femoral canal (non-diabeticBB Wistar rat) prior to Kirschner wire insertion and fracture.

Pack Boriding (Vanadium-Boron and Boron Control) Stainless Steel RodManufacturing:

During boriding of steel and other metallic and alloy surfaces, boronatoms diffuse into the material and form various types of metal borides.

A 1.6 mm Kirschner wire was annealed, cleaned and packed in a boridingpowder mixture contained within a 5 mm thick, heat resistant steel box.This allows the surfaces to be borided with a layer that is 10-20micrometers thick. A mixture was made consisting of boron carbide, VAC,silicon carbide, and a boriding activator. The parts conformed to thecontainer which they were packed, and then covered with a lid, whichrests inside the container. This container was then weighted with aniron slug to ensure even trickling of the boriding agent during themanufacturing. The container was then heated to the boriding temperaturein an electrically heated box with covered heating coils. The coatedrods were allowed to come to room temperature and wiped with 95% ethylalcohol prior to surgery for sterilization.

Vanadium Quantification in Animal Models

BB Wistar rats were anesthetized and confirmed to be non-responsive toexternal stimuli before beginning the surgical procedure. Theanesthetized rat was be exsanguinated by cardiac puncture using a 10 mlsyringe with a 22 gauge needle after shallow puncture just lateral tothe sternum and through the intercostal space. After puncturing thedermis and cardiac wall slight backpressure was placed on the plunger towithdraw blood from the ventricle. The collected blood was transferredto an appropriate container used for collection of plasma (heparinized)or serum (non-heparinized). Following the cardiac puncture, the ratswere euthanized via cervical dislocation.

The excised femora were be stripped of adhering muscle, tendon and othertissue after which the bones were rinsed three times with deionizedwater, and then placed on glassine paper and air dried. The pin wasrinsed once and stored in a clean conical tube. The liver, kidneys,brain, and left humerous collected were rinsed 3× and air dried. Theobjective of “drying” was to remove adhering water droplets after thewater rinse and to allow the true tissue weight to be recorded asprecisely as possible. The tissue's location on the glassine paper waschanged after a minute or two of air exposure. Air-drying does not lastlonger than 5 minutes. The dry bone is placed into a dry, previouslyacid-soaked/deionized-water rinsed, 7 ml pre-weighed sealablescintillation vial with plastic liner cap. Other organs were also storedin pre-rinsed, dry pre-weighed plastic vials of sizes appropriate foreach. The vials were labeled with an indelible marker indicating thedate of collection, right or left femur, Rat ID code, Investigator andStudy ID. The organs were then placed into a low temperature (−80degrees Celsius) freezer for storage until future analysis(quantification not currently planned).

The bone was carefully air-dried and any entrained fluid in theendosteal space drawn or shaken out to avoid bone weight anomaly.Beakers for subsequent collections were re-cleaned and femurs werehandled carefully to avoid cross contamination of specimens. Bones wereanalyzed via atomic absorption spectrophotometry to determine levels ofvanadium in bone, compared to standard levels in normal rat femoralbone. Analysis was based on a standard published technique 14 forquantifying vanadium levels in tissues.

Early-Stage Healing Analysis by Histomorphometry

The fractured femora were resected at 2, 4, 7, and 10 dayspost-fracture, decalcified, dehydrated, embedded in paraffin, andsectioned using standard histological techniques. Sections were stainedwith Masson's Trichrome (Accustain™ Trichrome Staining kit, SigmaDiagnostics, St. Louis, Mo.) for histological observation using anOlympus BH2-RFCA microscope (Olympus Optical Co., Ltd., Shinjuku-ku,Tokyo, Japan). Digital images were collected using a Nikon DXM1200Fdigital camera (Nikon, Tokyo, Japan). Cartilage, new bone, and totalcallus area were measured from the digital images using Image-Pro Plussoftware (version 5, Media Cybernetics, Inc., Silver Spring, Md.). Totalcartilage and new bone area were normalized to total callus area andexpressed as the percent area. Two independent reviewers were used tominimize inconsistencies.

Late-Stage Healing Analysis by Histomorphometry

To examine the effects of VAC at later stages of fracture healing,femora were resected from animals in the groups described above at days10, 14, and 21, embedded and sectioned using standard histologicaltechniques. This includes dehydration, soaking in Xylenes, and finallypre-embedding in a layer of Polymethylmethacrylate (PMMA). Afterembedding in pure PMMA and allowed to solidify in a hot water bath,slides were sectioned from the PMMA blocks, polished, and stained with acombination of Stevenel's blue and Van Gieson picro-fuchsin (SVG).Histological images of fracture calluses were obtained using an OlympusSZX12 upright microscope (Olympus Optical Co, LTD, Japan) connected viaa CCD camera (Optronics, Goleta, Calif.) to a personal computer andanalyzed with the Bioquant software package (Biometrics, Inc, Nashville,Tenn.). Parameters that were compared include a) callus area, b) percentcalcified tissue area, and c) percent cartilage area. Limitations ofthis procedure include production of slides with high thicknesses, dueto the difficulties associated with sectioning PMMA. This limits thenumber of possible sections that may be cut for staining in addition toanalysis of cellular morphology, due to overlapping layers of cells.

Early Immuohistochemistry

At days 2, 4, and 7, and 10, animals were injected intraperitoneallywith 30 mg/kg of 5-bromo-2′deoxyuridine (BrdU, Sigma Chemical Co., St.Louis, Mo.) one hour prior to sacrifice to label replicating cells as ameasure of cellular proliferation. The fractured femurs were resectedand fixed in formalin, decalcified (Immunocal, Decal Corp., Tallman,N.Y.), embedded in paraffin, and sectioned longitudinally (5 μm thick).Cells positive for BrdU incorporation were detected byimmunohistochemistry using commercially available reagents (DAKO Corp.,Carpentaria, Calif.). Digital images of each fracture were collectedwith an Olympus BH2-RFCA microscope equipped with a Nikon DXM1200fcamera. For each specimen, callus area was measured and the BrdUpositive cells in the periosteal callus region were counted using ImagePro Plus software. All BrdU positive cells in the external callus to amaximum of 1 cm proximal and distal of the fracture site and 3 mm fromthe external surface of the femur were counted. The number of BrdUpositive cells was normalized per unit area of callus and only one datumper rat (BrdU positive cells per mm2) was used for the statisticalanalysis.

Torsional Mechanical Testing

Torsional testing was conducted at weeks four and five using aservohydraulics machine (MTS Sys. Com., Eden Prairie, Minn.) with a 20Nm reaction torque cell (Interface, Scottsdale, Ariz.). Femurs weretested to failure at a rate of 2.0 deg/sec at four and six week timepoints. The peak torque, torsional rigidity, effective bulk modulus, andthe effective maximum shear stress (a) were determined with standardequations that model each femur as a hollow ellipse. In order to comparethe biomechanical parameters between different groups, the data wasnormalized by dividing each fractured femur value by its correspondingintact, contralateral femur value. Torsional mechanical testing islimited by differences in gauge length during bone potting in Field'smetal Placement and dimension of fracture gap can contribute to standarddeviations. Finally, this test is limited because it relies on amathematical model that assumes the femur is a hollow ellipse, asopposed to the natural architecture of femoral bone.

Data and Statistical Analysis

Analysis of variance (ANOVA) was performed followed by Holm-Sidakpost-hoc tests to determine differences between the treated VAC groupswith a group size larger than two. A Student's t-test was performed toidentify differences between the two treated groups in the VAC study(SigmaStat 3.0, SPSS Inc., Chicago, Ill.). A p value less than 0.05 wasconsidered statistically significant.

General Health of Animals

The age of the BB Wistar rats at the time of fracture surgery variedbetween 75 and 137 days. However, animals amongst treatment groups wereage and sex matched for each experiment. The percent weight changefollowing surgery to the day of sacrifice wars amongst treatment groups.

Results Vanadium Quantification in Animal Models

Locally injected VAC remains bound within the fractured femoraapproximately two weeks after local injection. These results weredetermined from the following experiment. Immediately prior to fracture,the femoral canal of each rat was injected with 0.1 ml of either salineor 4.35 mg/mL of VAC solution (4.35 mg/ml VAC solution; approximately1.5 mg VAC/kg weight of the rat; approximately 435 μg of VAC powder;approximately 84 μg of vanadium). To assess how rapidly the vanadiumdisperses from the fracture site, rats were sacrificed at one, four,seven, and 14 days after surgery to measure vanadium levels in thefracture callus. Atomic absorption spectrophotometry was used toquantify local vanadium levels and normalized compared to levels innormal rat femur bone. Significant differences (p<0.05) in localvanadium levels were detected between the right, fractured femora andleft, non-fractured femora of the rats treated with local vanadium atall time points examined (FIG. 8). The half-life of VAC is relativelyshort (6 days) according to Zhang et al and the quantity within thefractured femora significantly decreased at four, seven, and 14 dayscompared to the contralateral femora. At 14 days, the local level ofvanadium was significantly decreased (p<0.05) compared to days one,four, and seven.

Histomorphometric Analysis

In animals treated with VAC, histomorphometric analysis revealed astatistically higher (p<0.05) percent cartilage in 1.5 mg/kg VAC treatedfemora, compared to controls at both 7 and 10 days (Table 9). At 14days, percent mineralized tissue in both 1.5 mg/kg and 3 mg/kg VACtreated femora were significantly increased (p<0.05:1.5 mg/kg VAC,p<0.05: 3 mg/kg VAC) compared to saline controls (Table 9). After 21days, percent mineralized tissue was significantly increased (p<0.05) in1.5 mg/kg VAC treated femora. This VAC-mediated acceleration of healingmay be seen via histological sections at clays 10-21 (Table 9 FIG. 9).

TABLE 9 Late histology local VAC delivery without a carrier in normalrats 10 Days Post-Fracture 14 Days Post-Fracture Callus % Callus % AreaMineralized % Area Mineralized % (mm²) Tissue Cartilage (mm²) TissueCartilage Saline 15.8 ± 2.3 17.9 ± 5.2 16.3 ± 2.8 19.5 ± 4.8 15.6 ± 4.7 12.4 ± 4.9 Control (n = 5) (n = 5) (n = 5) (n = 6) (n = 6) (n = 6) 1.518.4 ± 3.6 15.8 ± 5.1  30.6 ± 12.4* 21.7 ± 5.0 21.3 ± 2.1* 14.2 ± 4.9mg/kg (n = 7) (n = 7) (n = 7) (n = 5) (n = 5) (n = 5) VAC 3.0 18.6 ± 2.915.2 ± 3.4 17.8 ± 5.5 19.3 ± 3.1 21.9 ± 3.2* 13.0 ± 6.4 mg/kg (n = 5) (n= 5) (n = 5) (n = 5) (n = 5) (n = 5) VAC 21 Days Post-Fracture Callus %Area Mineralized % (mm²) Tissue Cartilage Saline 20.0 ± 6.8 25.0 ± 6.16.1 ± 3.2 Control (n = 6) (n = 6) (n = 6) 1.5 20.1 ± 4.5  32.7 ± 2.9*11.0 ± 4.7  mg/kg (n = 5) (n = 5) (n = 5) VAC 3.0 20.3 ± 5.1 33.4 ± 5.29.4 ± 5.7 mg/kg (n = 4) (n = 4) (n = 4) VAC The data represent meanvalues (±S.D.). *Represents values statistically higher than salinecontrol, p < 0.05 versus saline control.

Early Immunohistochemistry

In animals treated with VAC, no significant differences in cellproliferation existed at two or four days post-fracture, butsignificantly more proliferating cells per unit area (p<0.05) wasobserved in the periosteum at seven and ten days post-fracture.

Mechanical Testing Local VAC Without a Carrier

The effect of local vanadium therapy on healing of femur fractures wasmeasured by torsional mechanical testing. At four weeks post-fracture,rats treated with vanadium displayed improved mechanical properties ofthe fractured femora compared to the untreated group. The maximum torqueto failure, torsional rigidity, maximum effective shear stress, andeffective shear modulus were all significantly increased compared to theuntreated group (p<0.05: 1.51 mg/kg VAC, p0.05: 3 mg/kg VAC) (Table 10).Radiographs taken at four weeks post-fracture support these mechanicaltesting results (FIG. 10). When the mechanical parameters of thefractured femora were normalized to the intact, contralateral femora,percent maximum torque to failure, percent torsional rigidity, andpercent effective shear modulus were still significantly greater in thelocal vanadium treated groups when compared to the saline control group(p<0.05: 1.5 mg/kg VAC, p<0.05: 3 mg/kg VAC). By five weekspost-fracture the maximum torque to failure and torsional rigidity weresignificantly greater in the 1.5 mg kg VAC heated group compared to bothcontrol and 3 mg/kg VAC groups respectively (p<0.05) (Table 11).

TABLE 10 Post-fracture mechanical testing of vanadium (VAC) in normalrats at 4-weeks Maximum Maximum Torsional Effective Torque to failureRigidity Effective Shear Shear Stress (Nmm) (Nmm²/rad) Modulus (MPa)(MPa) Control (n = 6)  161 ± 48  9,889 ± 4,719 258 ± 108 17 ± 4 0.25mg/kg  227 ± 64 28,218 ± 9,107 878 ± 416 25 ± 9 VAC (n = 6) 0.5 mg/kg   362 ± 49 *^(,#)   45,877 ± 13,079 * 1,107 ± 441    32 ± 13 VAC (n =6) 1.5 mg/kg    329 ± 117 *   34,526 ± 16,851 *  2,454 ± 2,370 *    69 ±59 * VAC (n = 6) 3.0 mg/kg    409 ± 43 *^(,#)   41,007 ± 11,236 *  2,948± 1,218 *     101 ± 18 *^(,#,&) VAC (n = 5) Fractured femur valuesnormalized to the contralateral (intact) femur Percent Percent Percentmaximum maximum Percent Effective Effective torque to failure torsionalrigidity Shear Modulus Shear Stress Control (n = 6)   27 ± 18   20 ± 10 4 ± 2 10 ± 5 0.25 mg/kg   49 ± 14    67 ± 21 * 14 ± 4  10 ± 3 VAC (n =6) 0.5 mg/kg    72 ± 19 *    103 ± 23 *^(,#) 16 ± 7   20 ± 11 VAC (n =6) 1.5 mg/kg   59 ± 28    76 ± 28 *   23 ± 12 *  26 ± 16 VAC (n = 6) 3.0mg/kg     79 ± 12 *    78 ± 10 *   20 ± 11 *    30 ± 12 *^(,#) (n = 5)The data represents average values ± standard deviation * Representvalues statistically higher than control, p < 0.05 versus control. ^(#)Represent values statistically higher than Extra Low Dose, p < 0.05versus Extra Low Dose. ^(&) Represent values statistically higher thanLowered Low Dose, p < 0.05 versus Lowered Low Dose.

TABLE 11 Post-fracture mechanical testing of VAC in normal rats at 5weeks Fractured femur values Maximum Maximum Effective Effective Torqueto Torsional Shear Shear failure Rigidity Modulus Stress (Nmm)(Nmm²/rad) (MPa) (MPa) Control  295 ± 164 20,111 ± 10,944 1,060 ± 693 45± 28 (n = 6) 1.5 mg/kg  471 ± 91*^(,#) 34,522 ± 8,347* 2,026 ± 924 75 ±26 VAC (n = 9) 3.0 mg/kg  335 ± 89  37,496 ± 12,846* 1.453 ± 683 43 ± 25VAC (n = 8) Fractured femur values normalized to the contralateral(intact) femur Percent Percent Percent maximum maximum Effective Percenttorque to torsional Shear Effective failure rigidity Modulus ShearStress Control  74 ± 42 80 ± 57   28 ± 29 31 ± 21 (n = 6) 1.5 mg/kg  99± 17 103 ± 33    39 ± 26 47 ± 25 VAC (n = 9) 3.0 mg/kg  64 ± 26 98 ± 28 23 ± 9 22 ± 11 VAC (n = 8) The data represents average value ± standarddeviation *Represent values shtistically higher than control, p < 0.05versus control. ^(#)Represent values statistically higher than highdose, p < 0.05 versus high dose.

Local VAC Without a Carrier in Diabetic Model

The effect of local vanadium therapy on healing of diabetic femurfractures was measured by torsional mechanical testing. Blood glucosewas monitored biweekly for type I diabetic BB Wistar rats andsubcutaneous Linplants™ (Linshin, Canada) were administered to alldiabetic animals, roughly every two weeks to maintain systemic glucoselevels. At six weeks post-fracture diabetic rats treated with vanadiumdisplayed significantly improved mechanical properties of the fracturedfemora compared to the untreated diabetic group. The maximum torque tofailure, torsional rigidity, maximum effective shear stress, andeffective shear modulus were all significantly increased compared to theuntreated diabetic group (p<0.05: 1.5 mg/kg VAC) (Table 12). When themechanical parameters of the fractured femora were normalized to theintact, contralateral femora, percent maximum torque to failure, percenttorsional rigidity, percent effective shear stress, and percenteffective shear modulus were still significantly greater in the localvanadium treated diabetic groups when compared to the untreated diabeticgroup (p<0.05: 1.5 mg/kg VAC). Torsional mechanical testing parametersfor the VAC treated diabetic animals were comparable to the non-diabeticanimals at six weeks.

TABLE 12 Post-fracture mechanical testing of non-diabetic, diabetic anddiabetic rats treated with VAC at 6-weeks Maximum Torque MaximumTorsional Effective Shear to failure (Nmm) Rigidity (Nmm²/rad) Stress(MPa) diabetic control (n = 23) 154 ± 69 475 ± 259 3 ± 2 1.5 mg/kg VACin  410 ± 71 *   43,089 ± 19,720 *    98 ± 53 *^(,#) diabetic (n = 3)normal (n = 12)  456 ± 66 *   33,784 ± 11,849 *  48 ± 16 * Normalized tocontralateral Percent maximum Percent maximum Percent Effective femurtorque to failure torsional rigidity Shear Stress diabetic control (n =23)  27 ± 10 27 ± 15 8 ± 4 1.5 mg/kg VAC in   85 ± 23 *   136 ± 111 *  33 ± 20 * diabetic (n = 3) normal (n = 12)   78 ± 15 * 86 ± 29   28 ±13 * The data represents average values ± standard deviation * Representvalues statistically higher than control, p < 0.05 versus control.^(#)Represent values statistically higher than normal, p < 0.05 versusnormal. The value of the numbers for diabetic control is obtained fromtwo papers of Gandhi (Insulin: Bone 2005: PRP:Bone 2006 and Beam et al2002 JOR). The value of 6-week normal group is an average of Gandhi'spaper and the investigator's 6 week mechanical test saline animals.

Local VAC/CaSO4 Formulations

When local vanadium with a calcium sulfate carrier was torsionallytested, results revealed significantly higher effective shear stress(p<0.05) for the 0.25 mg/kg VAC with calcium sulfate carrier group,compared to both the calcium sulfate buffer and 1.5 mg/kg VAC withcalcium sulfite carrier groups. When the mechanical parameters of thefractured femora were normalized to the intact, contralateral femora,maximum torque to failure, and percent effective shear modulus weresignificantly greater in the 0.25 mg/kg VAC with calcium sulfate carriergroup, compared to the calcium sulfate buffer group (p<0.05) (Table 13).

TABLE 13 Post-fracture mechanical testing of VAC/CaSO₄ in normal rats at4-weeks Maximum Torque Maximum Torsional Effective Effective to failureRigidity Shear Shear Stress (Nmm) (Nmm²/rad) Modulus (MPa) (MPa) Control(n = 6) 161 ± 48  9,889 ± 4,719 258 ± 108 17 ± 4 CaSO₄ Buffer  241 ± 17225,684 ± 20,795 680 ± 623  23 ± 16 (n = 9) 0.25 mg/kg    430 ± 133 *31,138 ± 11,518 1,178 ± 484 *     55 ± 21 *^(,#,&) VAC and CaSO₄ Carrier(n = 6) 1.5 mg/kg VAC  322 ± 157 26,302 ± 17,974 637 ± 395  29 ± 15 andCaSO₄ Carrier (n = 5) Fractured femur values normalized to thecontralateral (intact) femur Percent maximum Percent maximum PercentEffective Percent Effective torque to failure torsional rigidity ShearModulus Shear Stress Control (n = 6)  27 ± 18 20 ± 10 4 ± 2 10 ± 5 CaSO₄Buffer  37 ± 30 47 ± 47 7 ± 7  9 ± 6 (n = 9) 0.25 mg/kg VAC    85 ± 24*^(,#)  100 ± 49 *     24 ± 10 *^(,#,&) 18 ± 9 and CaSO₄ Carrier (n = 6)1.5 mg/kg VAC  64 ± 30 69 ± 47 15 ± 8  10 ± 7 and CaSO₄ Carrier (n = 5)The data represents average values ± standard deviation * Representvalues statistically higher than control, p < 0.05 versus control.^(#)Represent values statistically higher than CaSO₄ Buffer, p < 0.05versus CaSO₄ Buffer. ^(&)Represent values statistically higher than LowDose and CaSO₄ Carrier, p < 0.05 versus Low Dose and CaSO₄ Carrier.

Surface Modified VAC Coated Implants

Torsional mechanical testing of surface modified rods four weekspost-fracture demonstrated significantly greater maximum torque tofailure for the animals with vanadium-boron surface modified rodscompared to the group with untreated 316L stainless steel (SS) controlrods (p<0.05). When the mechanical parameters of the fractured femorawere normalized to the intact, contralateral femora, the percent maximumtorque to failure was significantly greater for the animals withvanadium-boron surface modified rods compared to the group withuntreated 316L stainless steel control rods (p<0.05). Although torsionalmechanical parameters were higher for the vanadium-boron surfacemodified rods, compared to the boron surface modified control rods, nosignificant differences were found between these groups (Tables 14 and15).

TABLE 14 Post-fracture mechanical testing on surface modifiedvanadium-bonded rods in normal rats (Pilot Data in Female Rats) at 4weeks Fractured femur values Maximum Maximum Maximum Torque to TorsionalShear Shear failure Rigidity Modulus Stress (Nmm) (Nmm²/rad) (MPa) (MPa)316 L Stainless Steel 178 ± 38  9,363 ± 5,032 235 ± 102 19 ± 3 ControlRod (n = 5) Boron Coated Control Rod 251 ± 93 19,683 ± 9,207 1,909 ±1,582  70 ± 46 (n = 3) 0.6 mg/kg Vanadium-Boron   305 ± 30 *   31,078 ±6,917 * 2,347 ± 1,649  60 ± 33 Coated Rod (n = 4) Fractured femur valuesNormalized to the contralateral (intact) femur Percent Percent Percentmaximum maximum Percent maximum torque to torsional shear shear failurerigidity modulus stress 316 L Stainless Steel   30 ± 18  19 ± 11 4 ± 211 ± 5 Control Rod (n = 5) Boron Coated Control Rod    68 ± 22 *  73 ±36 23 ± 18  33 ± 16 (n = 3) 0.6 mg/kg Vanadium-Boron   76 ± 9 *   107 ±36 *   38 ± 19 *   40 ± 20 * Coated Rod (n = 4) The data representsaverage values ± standard deviation * Represent values statisticallyhigher than control, p < 0.05 versus 316 L Stainless Steel controlgroup.

TABLE 15 Post-fracture medianical testing with sm-face modifiedvanadium- bonded rods in normal rats (Study in Male Rats) at 4 weeksMaximum Maximum Effective Effective Torque to Torsional Shear Shearfailure Rigidity, Modulus Stress (Nmm) (Nmm²/rad) (MPa) (MPa) 316 L 161± 48 9,889 ± 4,719 258 ± 108 17 ± 4 Stainless Steel Control Rod (n = 6)Boron Coated  269 ± 102 22,340 ± 12,323 400 ± 304 17 ± 8 Control Rod (n= 3) 0.6 mg/kg   366 ± 150 * 23,650 ± 11,718 609 ± 422  32 ± 15Vanadium-Boron Coated Rod (n = 5) Fractured femur values normalized tothe contralateral (intact) femur Percent Percent Percent Percent maximummaximum Effective Effective torque to torsional Shear Shear failurerigidity Modulus Stress 316 L  27 ± 18 20 ± 10 4 ± 2 10 ± 5 StainlessSteel Control Rod (n = 6) Boron Coated  45 ± 20 51 ± 34 7 ± 6 10 ± 6Control Rod (n = 5) 0.6 mg/kg   65 ± 25 * 52 ± 25 9 ± 5 19 ± 9 Vanadium-Boron Coated Rod (n = 5) The data represents average values ± standarddeviation * Represent values statistically higher than control, p < 0.05versus 316 L Stainless Steel control group.

Effect of VAC on Rats of Advanced Age

The effect of local vanadium therapy on healing of femur fractures innormal (non-diabetic) rats was measured by torsional mechanical testing.At four weeks post-fracture, fractured femurs from the rats of advancedage (190-195 days of age) treated with VAC had greater mechanicalproperties than the fractured femurs from the control group. When themechanical parameters of the fractured femora were non to the intact,contralateral femora, percent maximum torque to failure (saline groupvs. 1.5 mg/kg VAC group p<0.05 was significantly greater in the localvanadium treated groups when compared to the saline group (Table 16).

Healing was assessed by radiographic examination and quantified bymechanical testing. Local VAC treatment improved radiographic appearanceand significantly increased the mechanical strength of fractured femurs.At four weeks post-fracture, the average percent maximum torque tofailure of the fractured femora for 1.5 mg/kg VAC was significantly 76percent greater (44.0% of contralateral vs. 25.0%), compared to theuntreated saline group (Table 16), The data indicate that local VACtreatment enhanced bone regeneration during fracture healing even in apopulation of advanced age.

TABLE 16 Four weeks post-fracture mechanical testing with local vanadium(VAC) in rats of advanced age (Age: 190-195 days) Fractured Femur ValuesMaximum Maximum Effective Effective Mean Torque to Torsional Shear ShearAngle at Failure Rigidity Modulus Stress Failure (Nmm) (Nmm²/rad) (MPa)(MPa) (degrees) Saline Control 220 ± 76 4.2 × 10⁴ ± 1.7 × 10⁴ 2.2 × 10³± 1.5 × 10³ 36 ± 5  6 ± 3 (n = 3) 1.5 mg/kg VAC 324 ± 83 3.0 × 10⁴ ± 1.8× 10⁴ 1.3 × 10³ ± 1.3 × 10³  43 ± 17 12 ± 4 (n = 4) Fractured FemurValues Normalized to the Contralateral (Intact) Femur Percent PercentPercent Percent Maximum maximum Effective Effective Torque to TorsionalShear Shear Failure Rigidity Modulus Stress — Saline Control 2.5 ± 7 62± 19 24 ± 10 15 ± 7 NA (n = 3) 1.5 mg/kg VAC   44 ± 10 * 56 ± 37 16 ± 1816 ± 4 NA (n = 4) The data represents average values ± standarddeviation * Represents values statistically higher than saline control,p < 0.05 versus saline control. Student t-test between 2 groups

The effect of local vanadium therapy on healing of femur fractures innormal (non-diabetic) rats was measured by torsional mechanical testing.At four weeks post-fracture, fractured femurs from the rats treated withVAC had greater mechanical properties than the fractured femurs from thecontrol group, even if the VAC solution was autoclaved or gammairradiated prior to administration (FIG. 11, Table 17). For the 1.5mg/kg VAC group without sterilization, the maximum torque to failure(saline group vs. 1.5 mg/kg VAC group without sterilization p<0.05) andtorsional rigidity (saline group vs. 1.5 mg/kg VAC group withoutsterilization p<0.05) were significantly greater than the saline controlgroup. For the 1.5 mg/kg VAC autoclaved VAC group, the torsionalrigidity (saline group vs. 1.5 mg/kg autoclaved VAC group p<0.05) wassignificantly greater than the saline control group (Table 17). When themechanical parameters of the fractured femora were normalized to theintact, contralateral femora, percent torsional rigidity (saline groupvs. 1.5 mg/kg VAC group without sterilization p<0.05, saline group vs.1.5 mg/kg autoclaved VAC group p<0.05), and shear modulus (saline groupvs. 1.5 mg/kg VAC group without sterilization p<0.05) were significantlygreater in the local vanadium treated groups when compared to the salinegroup (Table 17).

TABLE 17 Four weeks post-fracture mechanical testing with local vanadium(VAC) without a Carrier Maximum Torque to Maximum Effective Effectivefailure Torsional Rigidity Shear Modulus Shear Stress (Nmm) (Nmm²/rad)(MPa) (MPa) Saline Control 161 ± 48  9.9 × 10³ ± 4.7 × 10³ 2.6 × 10² ±1.1 × 10² 17 ± 4 (n = 6) 1.5 mg/kg   329 ± 117 *   3.5 × 10⁴ ± 1.7 ×10⁴* 2.5 × 10³ ± 2.4 × 10³  69 ± 59 VAC without sterilization (n = 6)1.5 mg/kg 276 ± 79  2.6 × 10⁴ ± 3.7 × 10³ 7.9 × 10⁴ ± 9.4 × 10² 28 ± 4VAC Gamma Irradiated (n = 3) 1.5 mg/kg 292 ± 83   3.4 × 10⁴ ± 1.5 × 10⁴*9.6 × 10² ± 7.8 × 10² 26 ± 16 VAC Autoclaved (n = 5) Fractured FemurValues Normalized to the Contralateral (Intact) Femur Percent maximumPercent torque to Percent maximum Percent Effective Effective failuretorsional rigidity Shear Modulus Shear Stress Saline Control  27 ± 18 20 ± 10 4 ± 2 10 ± 5 (n = 6) 1.5 mg/kg   5 ± 78   76 ± 28*  23 ± 12* 26 ± 16 VAC without sterilization (n = 6) 1.5 mg/kg  50 ± 18  58 ± 10 9± 2 14 ± 5 VAC Gamma Irradiated (n = 3) 1.5 mg/kg  47 ± 12   70 ± 31* 15± 10 14 ± 7 VAC Autoclaved (n = 5) The data represents average values ±standard deviation *Represents values statistically higher than salinecontrol, p < 0.05 versus saline control. One-way ANOVA between 4 groupswith Hohn-Sidak post-hoc analysis.

Healing was assessed by radiographic examination and quantified bymechanical testing. Local VAC treatment improved radiographic appearanceand significantly increased the mechanical strength of fractured femurs.At four weeks post-fracture, the average percent maximum torsionalrigidity values for 1.5 mg/kg VAC without sterilization and followingthe autoclave process were significantly greater, with non-sterile VAC;2.8 times greater (76.0% of contralateral vs. 20.0%), and autoclavedVAC; 2.5 times greater (70.0% of contralateral vs. 20.0%) compared tothe saline control group. Percent shear modulus values for 1.5 mg/kg VACwithout sterilization was significantly greater; 4.8 times greater(23.0% of contralateral vs. 4.0%) compared to the saline control group.The data indicate that local VAC treatment enhanced bone regenerationduring fracture healing and indicates that effective sterilizationtechniques that may affect the stability and bioactivity of proteins, donot significantly alter the bioactivity of VAC.

The effect of local vanadium therapy on healing of femur fractures innormal (non-diabetic) rats was measured by radiographic analysis. Attwelve weeks post-fracture, fractured femurs from the rats treated withboth low (1.5 mg/kg) and high (3.0 mg/kg) VAC had no evidence of ectopichone formation, following resolution of the induced fracture (FIG. 12).Femora treated with local VAC demonstrated normal remodeling suggestingno evidence of a toxic/carcinogenic effect of VAC throughout fracturehealing. The above data have demonstrated an effective VAC therapeuticdosage range 0.5 to 3.0 mg/Kg, which resulted in two to three-foldincrease in mechanical parameter of torsion.

Comparison with Existing Therapies

The chart in FIG. 13 compares the vanadium technologies with thecurrently approved products (BMP-2 and Exogen) for fracture healing.Each of these studies examined the effectiveness of a therapeuticadjunct on femur fracture healing by measuring the maximum torque tofailure at the same timepoint (four weeks). Specifically the followingwere compared: (1) a single percutaneous dose (0.25 mg/kg) of VAC withthe calcium sulfate (CaSO₄) vehicle (red): (2) a single percutaneousdose (1.5 mg/kg) of VAC without a vehicle (blue); (3) a 316L stainlesssteel k-wire surface modified with vanadium (a process called vanadiumpack-boriding), implanted into the intramedullary canal of the femur(green); (4) BMP-2 study used a single percutaneous dose of BMP-2 (80μg) with buffer vehicle (orange); and (5) Exogen study used varyingexposure periods of ultrasound treatment (20 mins/day). The mosteffective duration (25 days) is shown in dark blue.

Thus, the results have demonstrated, among others, (a) that the use ofvanadium compounds (such as VAC) alone or as part of a formulation withan orthopedic carrier (CaSO4 for example) that is applied directly tothe site of fracture; and (b) that the use of orthopedic implants(pedicle screws, plates, rods, wires, etc.,) where the surface has beenmodified with vanadium via known thermal processing techniques. As aninsulin-mimetic adjunct, vanadium compounds can be used to acceleratebone regeneration by stimulating insulin signaling at the fracture site.Local VAC targets the beta-subunit of the insulin signaling receptor.The presence of the insulin mimetic also enhances cartilage andmineralized tissue formation. Our laboratory data demonstrated that VACtreatment significantly increases cell proliferation within thesubperiosteal region of the fracture callus (seven and ten dayspost-fracture). This translates into significantly higher percentcartilage within the fracture callus, (seven and ten dayspost-fracture). The percent mineralized tissue for local VAC treated ratanimal models was significantly higher than controls after 21 days. Thisaccelerated progression of the bone healing process results insignificantly enhanced mechanical testing parameters for VAC treatedanimals after four and five weeks, compared to controls.

Example 4 Insulin Mimetics Enhancing Spinal Fusion

Increased fusion rates were observed in a rat posterolateral lumbarspinal fusion model when treated with a time-released insulin implant incomparison with controls. The effects of insulin-mimetic agents wereanalyzed as an adjunct to spinal fusion in the rat posterolateral lumbarfusion model. Vanadyl acetylacetonate (VAC) or Zinc was made into apellet with Calcium Sulfate, and applied to the fusion bed withautograft in a rat posterolataeral lumbar fusion. These results werecompared with a control group treated with autograft and a palmitic acidpellet.

Study Design

The protocol was approved by the animal Institutional Care and UseCommittee at UMDNJ-New Jersey Medical School. Fifty skeletally matureSprague-Dawley rats weighing approximately 500 grams each underwentposterolateral intertransverse lumbar fusions with iliac crest mop-aftfrom L4-L5 utilizing a Wiltse-type approach. After exposure of thetransverse processes and high-speed burr decortication, one of fivepellets were added to the fusion site: a low dose Vanadium CalciumSulfate pellet (0.75 mg/kg), a high dose Vanadium Calcium Sulfate pellet(1.5 mg/kg), a low dose Zinc Calcium Sulfate pellet (0.5 mg/kg), a highdose Zinc Calcium Sulfate pellet (1.0 mg/kg), and a control ofmicro-recrystalized palmitic acid pellet. An equal amount of iliac crestautograft (approximately 0.3 g per side) was harvested and implantedwith each pellet. The rats were sacrificed at eight weeks, and spineswere harvested, removed of soft tissue, and tested by manual palpation,radiographs and MicroCT. All outcome parameters were independentlyreviewed by two separate individuals in a blinded manner and the lowergrade of fusion was accepted when there was a discrepancy.

Surgical Procedure

After obtaining general anaesthesia with intraperitoneal Ketamine (40mg/kg) and Xylazine (5 mg/kg), the lumbar region of the rat was shavedand cleansed with povidone iodine soaked gauze. A dorsal midlineincision was made from L3 to the sacrum. Two paramedian incisions weremade through the lumbar fascia 5 mm from the midline. Dissection wastaken to the iliac crest, and approximately 0.3 g of bone was harvestedwith small rongeurs. The harvested autograft was measured on a sterilescale in order to obtain 0.3 g per side. Blunt dissection was carrieddown posterolaterally, reflecting the paraspinal muscles lateral to thefacet joints on each side. The reflected paraspinal muscles were held inplace with retractors. The transverse processes of L4-L5 were cleaned ofsoft tissue, and decorticated with a high-speed burr (FIG. 14). Thecrashed autograft was then spread over and between the transverseprocesses at the appropriate level (L4-L5). An equivalent amount ofimplant, or blank was incorporated into the autograft bed (FIG. 15).Retractors were removed and the paraspinal muscles were allowed to coverthe fusion bed. The dorsal lumbar fascia was closed using a running 4-0resorbable suture and the skin was closed with interrupted 4-0resorbable sutures. The surgical site was treated with antibioticointment, and the rats were given a dose of Enrofloxacin antibiotic (10mg/kg). Radiographs were taken immediately after surgery. Blood glucoselevels were taken before surgery, and 12 and 24 hours after surgery. SeeTable 18.

TABLE 18 Systemic blood glucose levels (mg/dL) Before 12 24 Groupsurgery hours hours Controls 91.4 VAC-low 103.5 213.4 117.7 VAC-high102.9 153.2 90.7 Zn-low 106.0 122.8 101.8 Zn-high 109.3 120.0 89.0

Pellet Preparation

In order to prepare the pellets, 0.2 mL of each stock solution will bemixed with 0.4 g of CaSO₄ to obtain the appropriate consistency of thecarrier in a 1 mL syringe. It will then be injected into 2 mm diameterclear Tygon laboratory tubing and allowed to harden overnight

Once set, pellets will be sectioned into 7 mm pieces and autoclaved (tosterilize), prior to implantation.

Assumption: Weight of SD rat=0.45 kg

Vn Vn Zn Zn (0.75 mg/kg) (1.5 mg/kg) (0.5 mg/kg) (1.0 mg/kg) Mass of0.338 mg 0.675 mg 0.225 mg 0.45 mg treatment for each rat

In order o prepare the stock solution, the volume of solution in eachpellet will be calculated by using the volume ratio of solution tomixture.

Volume of CaSO₄ in each mixture

${D\mspace{14mu} {CaSO}_{4}} = {{2.96\mspace{14mu} g\text{/}{{cm}^{3}\left( {0.4{g\_ CaSO}_{4}} \right)}\text{/}\left( {2.96\frac{g}{{cm}^{3}}} \right)} = {{0.135\mspace{14mu} {cm}^{3}} = {0.135\mspace{14mu} {mL}}}}$

Volume of mixture and ratio

-   -   0.135 mL CaSO₄+0.2 mL solution=0.335 mL mixture    -   0.2 mL solution/0.335 mL mixture×100%=59.7% solution per unxture

Volume of each pellet, 1 mm radius, 7 mm height

-   -   V=πr²h,=π(1 mm)²(7 mm)=22 mm³=0.022 mL

Volume of solution in each pellet

-   -   0.022 mL×59.7%=0.0131 mL solution per pellet

Stock Solution (10 mL)

-   -   Because bilateral surgery is performed, mass of treatment (X)        must be halved for each pellet.

$\left( {\left( \frac{X}{2} \right)\text{/}0.0131\mspace{14mu} {mL}} \right) \times 10$

Vn Vn Zn Zn (0.75 mg/kg) (1.5 mg/kg) (0.5 mg/kg) (1.0 mg/kg) Mass of129.0 mg 258.0 mg 85.9 mg 171.8 mg treatment in each stock solution (10ml)

Radiographic Analysis

Posteroanterior radiographs at 35 kV for 90 seconds were taken at eightweeks after sacrifice and harvest. All soft tissue was removed prior toradiographic exam. Two blinded independent observers graded theradiographs as solid fusion mass bilaterally (A), unilateral fissionmass (B), small fission mass bilaterally (C), and graft resorption (D),based on previously published radiographic scales. See Table 19.

TABLE 19 Radiographs Group A B C D Kappa P Value Controls (n = 9) 2 3 13 0.297 VAC-low (n = 10) 3 3 0 4 0.583 0.807 VAC-high (n = 10) 5 3 1 10.667 0.270 Zn-low (n = 10) 7 1 2 0 0.512 0.066 Zn-high (n = 10) 7 3 0 01.0 0.050 A = solid fusion mass bilaterally B = unilateral fusion mass C= small fusion mass bilaterally D = Graft resorption

Based on radiographs (FIG. 16), in the high dose vanadium group 5/10 hadsolid fusion mass bilaterally, 3/10 had unilateral fusion, 1/10 hadsmall fusion mass bilaterally, and 1/10 had graft resorption. The lowdose vanadium group had 3/10 solid fusion mass bilaterally, 3/10 hadunilateral fusion, 0/10 had small fusion mass bilaterally, and 4/10 hadgraft resorption. The high dose zinc, group had 7/10 solid fusion massbilaterally, 3/10 had unilateral fusion, 0/10 had small fusion massbilaterally, and 0/10 had graft resorption. The low dose zinc group had7/10 solid fusion mass bilaterally, 1/10 had unilateral fusion, 2/10 hadsmall fusion mass bilaterally, and 0/10 had graft resorption. Thecontrol group had 2/9 solid fusion mass bilaterally, 3/9 unilateralfusion, 1 small fusion mass bilaterally, and 3/9 had graft resorption.

Manual Palpation

After removal of all soft tissue, two blinded independent observersmanually palpated and stressed across the fusion site (L4-L5). Specimenswere graded as fused (A), partially fused (B), and not fused (C). SeeTable 20.

TABLE 20 Manual palpation Group A B C Kappa P Value Controls (n = 9) 0 18 0.156 VAC-low (n = 10) 1 4 5 0.130 0.072 VAC-high (n = 10) 6 2 2 0.4120.002 Zn-low (n = 10) 3 4 3 0.565 0.055 Zn-high (n = 10) 4 1 5 0.3060.008 A = fused B = partially fused C = not fused

Based on manual palpation in the high dose Vanadium group 6/10 had solidfusion, 2/10 were partially fused, and 2/10 were not fused. In the lowdose vanadium group, 1/10 had solid fusion, 4/10 were partially fused,and 5/10 were not fused. In the high dose Zinc group, 4/10 had solidfusion, 1/10 had partially fused, and 5/10 were not fused. In the lowdose Zinc group, 3/10 had solid fusion, 4/10 had partially fused, and3/10 were not fused. In the control group, 0/9 had solid fusion, 1/9 hadpartially fused, and 8/9 were not fused.

Micro CT Analysis

TABLE 21 MicroCT Mean Bone Std Group Volume mm³ Dev P value Table 21aANOVA p = 0.006 Vn high dose (n = 10) 170.8 37.1 <0.01 vs control Vn lowdose (n = 10) 167.4 23.5 <0.5 vs control Controls (n = 9) 126.7 26.3Table 21b ANOVA p = 0.002 Zn high dose (n = 10) 172.7 26.4 <0.01 vscontrol Zn low dose (n = 10) 172.9 31.6 <0.5 vs control Controls (n = 9)126.7 26.3

Based on MicroCT analysis, the mean bone volume of the L4/L5 transverseprocesses and fusion mass for controls was 126.7 mm³. In the high doseVanadium group there was 170.8 mm³, and in the low dose Vanadium groupthere was 167.4 mm³. The high dose Zinc group had a mean of 172.7 mm³,and the low dose Zinc group had a mean of 172.9mm³. Differences betweeneach experimental group versus controls were significant (see Table 21).

Statistical Analysis

A Mann-Whitney Rank Test was performed for analysis of radiographs andmanual palpation. Kappa values were calculated for inter-rateragreement. ANOVA was performed for amt of new bone limitation as permicro CT with secondary test using Holm Sidak test. Statistical analysiswas performed using SigmaStat.

Of the 50 animals, one of the control rats died on postoperative dayone, likely due to anaesthesia. The remaining 49 rats had nocomplications and were sacrificed as planned (0.02% perioperativemortality rate).

The foregoing examples and description of the preferred embodimentsshould be taken as illustrating, rather than as limiting the presentinvention as defined by the claims. As will be readily appreciated,numerous variations and combinations of the features set forth above canbe utilized without departing from the present invention as set forth inthe claims. Such variations are not regarded as a departure from thespirit and script of the invention, and all such variations are intendedto be included within the scope of the following claims. All referencescited hereby are incorporated by reference in their entirety.

1-12. (canceled)
 13. A bone regeneration material for bone fusion or void filling, comprising an osteoconductive carrier and an active ingredient consisting of a therapeutically effective amount of an insulin-mimetic zinc or manganese compound.
 14. The material according to claim 13, wherein said osteoconductive carrier is selected from the group consisting of autograft material, allograft material, calcium phosphate, calcium sulfate, xenograft materials, alloplastic graft biomaterials, and orthopedic biocomposites.
 15. An orthopedic or spinal implant, comprising at least one bone-contacting surface comprising an active ingredient consisting of an insulin-mimetic, zinc or manganese compound or composition thereof.
 16. The orthopedic or spinal implant of claim 15, wherein the orthopedic implant is selected from the group consisting of screws, plates, rods, k-wires, pins, hooks, anchors, intramedullary devices, pedicle screws, pedicle hooks, spinal fusion cages, spinal fusion plates, prostheses, and porous metal.
 17. The orthopedic or spinal implant of claim 16, wherein said implant is made from a metal selected from the group consisting of titanium, alloys thereof, tantalum, alloys thereof, cobalt chrome alloys, steel alloys, and combinations thereof.
 18. The orthopedic or spinal implant of claim 17, wherein said steel alloy is a stainless steel.
 19. The orthopedic or spinal implant of claim 16, wherein said implant is made from a polymeric material.
 20. The orthopedic or spinal implant of claim 19, wherein said polymeric material comprises a polymer selected from polyglycolic acid (PGA), poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), polycaprolactone (PCL), polyether ether ketone (PEEK), polyethylene terephthalate (PET), polypropylene (PP), polycarbonates (PC), poly(ortho esters) (POEs), and combinations thereof.
 21. A method of enhancing spinal fusion in a spinal fusion surgical procedure, the method comprising the steps of: exposing a portion of each of adjacent vertebrae; and placing the bone regeneration material of claim 13 within an area between the exposed portions of the adjacent vertebrae and in contact with the exposed portions of both vertebrae, wherein said osteoconductive carrier of said bone regeneration material comprises a supplementary bone tissue material or synthetic bone void filler; wherein the insulin-mimetic zinc or manganese compound is in an amount effective to increase the rate of fusion of the two vertebrae with the bone tissue material.
 22. The method according to claim 21, wherein said bone tissue material or said bone void filler is combined with said insulin mimetic zinc or manganese compound in a formulation suitable for implantation.
 23. The method according to claim 21, wherein said bone regeneration material further comprises a surgically acceptable carrier.
 24. The method of claim 23, characterized in that said bone regeneration material is a calcium sulfate pellet comprising said insulin-mimetic zinc or manganese compound.
 25. The method according to claim 21, wherein said supplementary bone tissue is selected from the group consisting of autograft bone tissue, allograft bone tissue, xenograft bone tissue, and orthopedic biocomposite material.
 26. The method of claim 21, wherein the synthetic bone void filler is a ceramic bone-graft substitute.
 27. The method of claim 25, wherein the method is posterior fusion, said exposing step comprises decortication of the native host bone of the lamina or lateral masses to form a prepared area, and said placing step comprises packing a bone grafting mixture comprising the supplementary bone tissue material or a synthetic bone void filler and said insulin-mimetic zinc or manganese compound over the prepared area to induce segmental fusion.
 28. The method of claim 25, wherein the method is posterolateral fusion, said exposing step comprises decortication of the side of the facet joints and one or more transverse processes to form a prepared area, and said placing step comprises packing a bone grafting mixture comprising the supplementary bone tissue material or a synthetic bone void filler and said insulin-mimetic zinc or manganese compound over the prepared area to induce segmental fusion.
 29. The method according to claim 21, wherein said placing step comprises implanting an interbody device with a central chamber packed with said supplementary bone tissue material or said synthetic bone void filler and said insulin-mimetic zinc or manganese compound to enhance fusion between the vertebral bodies of the anterior column of the spine.
 30. The method of claim 29, wherein said placing step is performed after an exposing step comprising an anterior discectomy or an anterior decompression or an anterior corpectomy. 